我们感谢王大卫，阿纳托利Urisman，艾米奇石，Kael菲舍尔，邓健泓，和亚历山大Greninger的初步发展和优化的Virochip协议。这项工作是支持K08授予美国国立卫生研究院和雅培发现奖（消委会）和霍华德休斯医学研究所（JLD）。

Virochip检测的步骤包括:（1）从临床标本的核酸提取，（2）提取的RNA 逆转录和第二链cDNA合成，（3）随机引物的cDNA PCR扩增，（3）Cy3标记的荧光染料掺入（4）杂交标记的材料Virochip芯片，（5）扫描和分析（图1）。如下图所示的协议，将展示从儿童流感样病例鼻拭子样品的Virochip检测上使用。 引物序列引物5&#39; - GTTTCCCAGTCACGATA（N 9）-3" 引物乙5&#39; - GTTTCCCAGTCACGATA - 3" 1。核酸提取<ol><li>从临床标本的核酸提取应执行2级生物安全（BSL - 2）或更高的条件下，这些样本有潜在的传染性。 </li><li>鼻拭子样本运送病毒控股中期。分装200μL的样品至1.5毫升的Eppendorf离心管。 </li><li> 可选:通过样品通过0.22微米的过滤器（净化病毒颗粒） </li><li> 可选:用DNase（降解宿主基因组DNA和净化保护，encapsidated病毒核酸每制造商的说明使用的Turbo DNA酶试剂盒（Ambion公司）的预处理，样品） </li><li>提取的样品使用Zymo ZR的病毒RNA提取试剂盒或每Qiagen公司Ultrasens病毒试剂盒制造商的指示。 </li></ol> 2。逆转录和第二链cDNA合成（"回合一个"） <ol><li> 1 UL 40 pmol /μL底漆4 UL提取RNA。加热至65 °让C为5分钟在一个热循环仪（例如GeneAmp PCR系统9700），然后在室温× 5分钟冷却。 </li><li>添加5μL2微升5X RT缓冲液，1μL12.5毫米的dNTP，1μL水，0.5μL0.1M数码地面电视，0.5μL，SSIII RT（Invitrogen公司）组成的一个主混合。在42 ° C孵育× 60分钟。 </li><li>在一个可编程的热循环，加热至94 ° C X 2分钟（中止逆转录反应），冷却至10 ° C，脉冲离心机，然后加入5μLSequenase 1μL5X Sequenase缓冲液，3.8μL水组成的混合，和0.15μLSequenase（USB公司）。 第二链的合成，斜10 ° C至37 ° C，超过8分钟，保持8分钟，然后加热至94 ° C X 2分钟（灭活Sequenase），冷却至10 ° C（按住）。 </li><li>路可选 :一个样品的cDNA在这一点上可以存储在-20 ° C。 </li></ol> 3。随机引物PCR扩增的cDNA（"B轮"） <ol><li> 5路UL A样品添加45μL5μL的10X PCR缓冲液，1μL12.5毫米的dNTP，1μL100 pmol /μL引物b，1μL，KlenTaq LA（Sigma公司），以及37μL水组成的混合主。 </li><li>运行PCR协议如下:94 ° C，2分钟→（25周期94℃，30 S / 50 ° C，45秒/ 72 ° C，1分钟）→72 ° C，5分钟→10 ° C（持有） </li><li>路B瓶检查在1.5％琼脂糖凝胶电泳凝胶。涂片从约200 - 1000 BP应可视化（图2A） </li></ol> 4。 Cy3标记荧光染料法团<ol><li> 可选 :另一样品可与Cy5的荧光染料标记相同的芯片杂交。 </li><li>要结合AA -的dNTP，添加5号B瓶UL UL主组成的混合5 UL 10X PCR缓冲液，1 UL 12.5毫米AA -的dNTP（Invitrogen公司），1 UL 100 pmol / UL引物b，1至45， 1 UL KlenTaq洛杉矶（Sigma公司），和37 uL的水。运行PCR协议如下:94 ° C，2分钟→（15周期94℃，30 S / 50 ° C，45秒/ 72 ° C，1分钟）→72 ° C，5分钟→10 ° C（持有） </li><li>清洁道中的样本DNA清洁和每制造商的指示选矿厂5包（Zymo研究）。洗脱10微升。 </li><li>新增1 UL 1M碳酸氢钠（Sigma）和1 UL Cy5的（通用电气医疗集团）号彗星样本。 </li><li>孵育1个小时在黑暗中。 </li><li>清洁Cy3标记的样本DNA清洁和每制造商的指示选矿厂5包（Zymo研究）。洗脱12微升。 </li></ol> 5。杂交Virochip芯片<ol><li> 可选:使用1.5μL检查cDNA的浓度和染料的金额纳入上Nanodrop分光光度计（样本正常化） </li><li>在PCR地带管添加1μL的25X碎片缓冲区，5μL的5倍封闭液，9.5μLCy3标记，标记的样品（或金额正常化），和水的总体积至25μL（安捷伦） </li><li>加入25μL2X GEX杂交缓冲液（安捷伦）。离心简单，赶走气泡。 </li><li>加载到垫片幻灯片40μL的样品。 </li><li>广场安捷伦印Virochip阵列（Californi大学旧金山/安捷伦）（标有"安捷伦"下来方）垫片幻灯片上并拧紧螺钉紧。 </li><li>杂交一夜之间在65 ° C烘箱，在10转的速度设置。 </li><li>清洗阵列，预热缓冲区2（安捷伦）至37 ° C。除了在缓冲区1（安捷伦）垫片幻灯片/阵列夹心水下。在缓冲区1 × 1分钟洗净。洗，在预热的缓冲2 × 1分钟。慢慢地从缓冲区2中删除的幻灯片，要小心，以避免气泡（使用kimwipe到灯芯掉多余的水分，要小心，以避免直接接触阵列的主动方） </li><li>成幻灯片固定放置幻灯片和插入阵列扫描仪。 </li></ol> 6。 Virochip扫描和分析<ol><li>扫描Cy3标记在5微米或根据仪器的标准扫描协议（图2B）2微米阵列。 </li><li>通过目测，聚类分析，或自动化Virochip分析程序分析Virochip芯片，电子预测11。这三种方法，流感病毒很容易发现在鼻拭子样本（从流感样病例的孩子）（图2C） </li></ol> 7。代表性的成果: 是正确的协议时，应看到涂片，在琼脂糖凝胶电泳后的路乙一步（图2A）。安捷伦平台上Virochip芯片将难以直观分析（图2B），但如果病毒是目前应随时确定目视检查，聚类分析，和/或E -预测（图1C）。样品可以添加与测量，从已知的病毒（如烟草花叶病毒的MS2噬菌体）核酸作为一个检测的阳性对照的数量。 <img src="/files/ftp_upload/2536/2536fig1.jpg" alt="图1" /> 图1。随机扩增示意图Virochip芯片处理和分析 ，从临床标本中提取核酸，用荧光染料标记，杂交Virochip芯片。在2微米分辨率的微阵列扫描和使用各种计算工具，包括电子预测分析。 <img src="/files/ftp_upload/2536/2536fig2.jpg" alt="图2" /> 图2。在Virochip检测的步骤后随机，涂抹200 PCR扩增- 1000 BP可以用凝胶电泳观察（一）（二 ）三个Virochip微阵列8阵列/玻片上显示，与一个小区域。一吹，在右下角的插图芯片（C）使用E自动芯片病毒分析预测揭示了A型流感病毒的临床样本的存在。很高的相似性得分（S = 0.55）对应的p值接近于零，从而使这一极其显着的预测。

<ol>
	<li>Wang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray-based+detection+and+genotyping+of+viral+pathogens.">Microarray-based detection and genotyping of viral pathogens.</a> Proc Natl Acad Sci U S A. 99, 15687-15692 (2002).</li><li>Chiu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+of+a+critical+respiratory+illness+caused+by+human+metapneumovirus+by+use+of+a+pan-virus+microarray.">Diagnosis of a critical respiratory illness caused by human metapneumovirus by use of a pan-virus microarray.</a> J Clin Microbiol. 45, 2340-2343 (2007).</li><li>Chiu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray+detection+of+human+parainfluenzavirus+4+infection+associated+with+respiratory+failure+in+an+immunocompetent+adult.">Microarray detection of human parainfluenzavirus 4 infection associated with respiratory failure in an immunocompetent adult.</a> Clin Infect Dis. 43, e71-e76 (2006).</li><li>Chiu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utility+of+DNA+microarrays+for+detection+of+viruses+in+acute+respiratory+tract+infections+in+children.">Utility of DNA microarrays for detection of viruses in acute respiratory tract infections in children.</a> J Pediatr. 153, 76-83 (2008).</li><li>Kistler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pan-viral+screening+of+respiratory+tract+infections+in+adults+with+and+without+asthma+reveals+unexpected+human+coronavirus+and+human+rhinovirus+diversity.">Pan-viral screening of respiratory tract infections in adults with and without asthma reveals unexpected human coronavirus and human rhinovirus diversity.</a> J Infect Dis. 196, 817-825 (2007).</li><li>Rota, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+novel+coronavirus+associated+with+severe+acute+respiratory+syndrome.">Characterization of a novel coronavirus associated with severe acute respiratory syndrome.</a> Science. 300, 1394-1399 (2003).</li><li>Wang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+discovery+and+sequence+recovery+using+DNA+microarrays.">Viral discovery and sequence recovery using DNA microarrays.</a> PLoS Biol. 1, e2-e2 (2003).</li><li>Urisman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+novel+Gammaretrovirus+in+prostate+tumors+of+patients+homozygous+for+R462Q+RNASEL+variant.">Identification of a novel Gammaretrovirus in prostate tumors of patients homozygous for R462Q RNASEL variant.</a> PLoS Pathog. 2, 25-25 (2006).</li><li>Kistler, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+of+divergent+avian+bornaviruses+from+cases+of+proventricular+dilatation+disease:+identification+of+a+candidate+etiologic+agent.">Recovery of divergent avian bornaviruses from cases of proventricular dilatation disease: identification of a candidate etiologic agent.</a> Virol J. 5, e25-e25 (2008).</li><li>Chiu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+cardioviruses+related+to+Theiler's+murine+encephalomyelitis+virus+in+human+infections.">Identification of cardioviruses related to Theiler's murine encephalomyelitis virus in human infections.</a> Proc Natl Acad Sci U S A. 105, 14124-14129 (2008).</li><li>Urisman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=E-Predict:+a+computational+strategy+for+species+identification+based+on+observed+DNA+microarray+hybridization+patterns.">E-Predict: a computational strategy for species identification based on observed DNA microarray hybridization patterns.</a> Genome Biol. 6, R78-R78 (2005).</li></ol>

此处所述的Virochip协议是复杂的，需要细致和熟练的科研技术人员。正确的试剂浓度和PCR，标签条件，和杂交是至关重要的。 Virochip协议可以很容易地修改，以适应分析病变组织的组织提取方法，如TRIZOL（Invitrogen）的核酸提取使用。探头可以在必要时添加或删除，以获得所需的频谱和覆盖的广度，例如，可以设计和探针检测的细菌和真菌，如非病毒的目标"飞"的补充。 Virochip检测目前正在开发的一些应用，包括在临床实验室，疫情调查，纯度的药物和疫苗的筛选，和新的病毒病原发现的广谱病毒诊断。

<table border="1"><tbody><tr><th>试剂/材料名称</th><th>试剂/材料名称</th></tr><tr><td>西甲</td><td> 5&#39; - GTTTCCCAGTCACGATA（N9）-3" </td></tr><tr><td>引物b </td><td> 5&#39; - GTTTCCCAGTCACGATA - 3&#39; </td></tr><tr><td rowspan="5"> RT的master mix（5微升） </td><td> 2μL，5X RT缓冲</td></tr><tr><td> 1μL，12.5毫米的dNTP（Invitrogen公司） </td></tr><tr><td> 1μL水</td></tr><tr><td> 0.5微升0.1M数码地面电视</td></tr><tr><td> 0.5μL，SSIII RT（Invitrogen公司） </td></tr><tr><td rowspan="3"> Sequenase混合（5微升） </td><td> 1μL5X Sequenase缓冲区</td></tr><tr><td> 3.8μL水</td></tr><tr><td> 0.15微升Sequenase（USB公司） </td></tr><tr><td rowspan="5">路乙PCR扩增预混试剂（45微升） </td><td> 5μL的10X PCR缓冲液</td></tr><tr><td> 1μL，12.5毫米的dNTP（Invitrogen公司） </td></tr><tr><td> 1μL，100 pmol /μL的引物b </td></tr><tr><td> 1μLKlenTaq洛杉矶（Sigma公司） </td></tr><tr><td> 37μL水</td></tr><tr><td rowspan="5">道中PCR的master mix（45微升） </td><td> 5 UL 10X PCR缓冲液</td></tr><tr><td> 1μL，12.5毫米的dNTP（Invitrogen公司） </td></tr><tr><td> 1μL，100 pmol /μL的引物b </td></tr><tr><td> 1μLKlenTaq洛杉矶（Sigma公司） </td></tr><tr><td> 37μL水</td></tr><tr><td rowspan="4">杂交缓冲液（25微升） </td><td> 1μL25X碎片缓冲液（安捷伦） </td></tr><tr><td> 5μL的5倍封闭液（安捷伦） </td></tr><tr><td> Cy3标记，标记的样品9.5μL（或金额正常化） </td></tr><tr><td>加水到总体积25μL </td></tr><tr><td>安捷伦Virochip芯片</td><td>待许可证;邱实验室，加州大学旧金山分校</td></tr></tbody></table>

using a pan-viral microarray assay (virochip) to screen clinical samples for viral pathogens

该病毒会导致许多传染病的诊断，由于固有的病毒序列的多样性以及持续出现新型病毒的病原体，如SARS冠状病毒和2009年H1N1新型流感病毒，是不是用传统的方法检测，很难。为了应对这些挑战，我们以前开发和验证一个泛病毒的基因芯片平台与检测能力上的保守序列的同源性1的基础上所有已知的病毒以及新颖变种Virochip。使用的Virochip，我们已经确定了相关病毒与呼吸道感染，包括原因不明的重大疾病住院的患者案件，灵敏度相当于或优于传统的临床试验 2-5，全方位。 Virochip也被用来确定新的病毒，包括SARS冠状病毒6,7，一种新型的鼻病毒分支5，XMRV病毒（逆转录病毒与前列腺癌）8，禽流bornavirus（消耗性疾病在鹦鹉的原因）9 ，小说和儿童呼吸道疾病和腹泻病10 cardiovirus。 Virochip目前的版本已经被移植到安捷伦的芯片平台，并根据截至2009年12月从GenBank中的病毒超过1500〜〜36000探头组成。在这里，我们展示了在处理Virochip检测从开始涉及的步骤来完成（〜24小时的周转时间），包括样品核酸的提取，使用随机引物的PCR扩增，荧光染料掺入，与芯片杂交，扫描，并分析。

Watch this Scientific Journal Video about Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens at JoVE.com

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

Virochip是一个泛病毒的芯片设计，同时检测保守序列同源性的基础上所有已知的病毒，以及新颖的病毒。在这里，我们演示了如何运行的Virochip检测，分析存在的已知和未知病毒的临床样本。

使用泛病毒基因芯片检测（Virochip）屏幕病毒病原体的临床标本

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

using-pan-viral-microarray-assay-virochip-to-screen-clinical-samples

Research

JoVE Journal

Immunology and Infection

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

18.8K 次观看.  University of California, San Francisco. Virochip是一个泛病毒的芯片设计，同时检测保守序列同源性的基础上所有已知的病毒，以及新颖的病毒。在这里，我们演示了如何运行的Virochip检测，分析存在的已知和未知病毒的临床样本。

视频: Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

Determining the Phagocytic Activity of Clinical Antibody Samples

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as pathology of disease. While the properties of the variable domains of antibodies have long been considered critical to in vivo function, the ability of antibodies to recruit innate immune cells via their Fc domains has become increasingly appreciated as a major factor in their efficacy, both in the setting of recombinant monoclonal antibody therapy, as well as in the course of natural infection or vaccination1-3.
Importantly, despite its nomenclature as a constant domain, the antibody Fc domain does not have constant function, and is strongly modulated by IgG subclass (IgG1-4) and glycosylation at Asparagine 2974-6. Thus, this method to study functional differences of antigen-specific antibodies in clinical samples will facilitate correlation of the phagocytic potential of antibodies to disease state, susceptibility to infection, progression, or clinical outcome.
Furthermore, this effector function is particularly important in light of the documented ability of antibodies to enhance infection by providing pathogens access into host cells via Fc receptor-driven phagocytosis7. Additionally, there is some evidence that phagocytic uptake of immune complexes can impact the Th1/Th2 polarization of the immune response8.
Here, we describe an assay designed to detect differences in antibody-induced phagocytosis, which may be caused by differential IgG subclass, glycan structure at Asn297, as well as the ability to form immune complexes of antigen-specific antibodies in a high-throughput fashion. To this end, 1 &mu;m fluorescent beads are coated with antigen, then incubated with clinical antibody samples, generating fluorescent antigen specific immune complexes. These antibody-opsonized beads are then incubated with a monocytic cell line expressing multiple Fc&gamma;Rs, including both inhibitory and activating. Assay output can include phagocytic activity, cytokine secretion, and patterns of Fc&gamma;Rs usage, and are determined in a standardized manner, making this a highly useful system for parsing differences in this antibody-dependent effector function in both infection and vaccine-mediated protection9.

We present a high-throughput flow cytometric assay to determine the phagocytic activity of antigen-specific antibodies from clinical samples, utilizing fluorescent antigen-coated beads and a monocytic cell line expressing multiple Fc receptors&#x2014;providing receptor usage and phagocytic activity determinations in a standardized and reproducible fashion for any antigen of interest.

确定临床抗体样品的吞噬活性

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as ...

科研

免疫与感染

Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many advantages for ease and standardization of immunological assessments, but detrimental effects of this process have been observed on some cell subsets, such as granulocytes, B cells, and dendritic cells 1-3. Assaying fresh leukocytes gives a more accurate picture of the in vivo state of the cells, but is often difficult to perform in the context of large clinical trials. Fresh cell assays are dependent upon volunteer commitments and timeframes and, if time-consuming, their application can be impractical due to the working hours required of laboratory personnel. In addition, when trials are conducted at multiple centers, laboratories with the resources and training necessary to perform the assays may not be located in sufficient proximity to clinical sites. To address these issues, we have developed an 11-color antibody staining panel that can be used with Trucount tubes (Becton Dickinson; San Jose, CA) to phenotype and enumerate the major leukocyte populations within the peripheral blood, yielding more robust cell-type specific information than assays such as a complete blood count (CBC) or assays with commercially-available panels designed for Trucount tubes that stain for only a few cell types. The staining procedure is simple, requires only 100 &mu;l of fresh whole blood, and takes approximately 45 minutes, making it feasible for standard blood-processing labs to perform. It is adapted from the BD Trucount tube technical data sheet (<a href="http://www.bdbiosciences.com/external_files/is/doc/tds/Package_Inserts_CE/live/web_enabled/23-3483-06_PI_Trucount tubes_CE_EN.pdf" target="_blank">version 8/2010</a>). The staining antibody cocktail can be prepared in advance in bulk at a central assay laboratory and shipped to the site processing labs. Stained tubes can be fixed and frozen for shipment to the central assay laboratory for multicolor flow cytometry analysis. The data generated from this staining panel can be used to track changes in leukocyte concentrations over time in relation to intervention and could easily be further developed to assess activation states of specific cell types of interest. In this report, we demonstrate the procedure used by blood-processing lab technicians to perform staining on fresh whole blood and the steps to analyze these stained samples at a central assay laboratory supporting a multicenter clinical trial. The video details the procedure as it is performed in the context of a clinical trial blood draw in the HIV Vaccine Trials Network (HVTN).

In this report, we demonstrate the staining and analysis steps of a phenotyping assay performed on fresh whole blood to enumerate major innate and adaptive leukocyte populations. We emphasize considerations for performing these procedures in the context of a multicenter clinical trial.

枚举主要外周血白细胞人口多中心临床试验，使用全血表型分析

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many ...

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 ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

使用点击化学测量的影响，病毒感染宿主细胞RNA合成

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

利用荧光蛋白监视非洲锥虫Glycosome动态

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the natural reservoir for IAV, but IAV can cross the species barrier to poultry, swine, horses and humans. Avian viruses recognize sialic acid attached to a penultimate galactose by a &#945;2-3 linkage (avian-type receptors) whereas human viruses preferentially recognize sialic acid with a &#945;2-6 linkage (human-type receptors). To monitor if avian viruses are adapting to human type receptors, several methods can be used. Glycan microarrays with diverse libraries of synthetic sialosides are increasingly used to evaluate receptor specificity. However, this technique is not used for measuring avidities. Measurement of avidity is typically achieved by evaluating the binding of serially diluted hemagglutinin or virus to glycans adsorbed to conventional polypropylene 96-well plates. In this assay, glycans with &#945;2-3 or &#945;2-6 sialic acids are coupled to biotin and adsorbed to streptavidin plates, or are coupled to polyacrylamide (PAA) which directly adsorb to the plastic. We have significantly miniaturized this assay by directly printing PAA-linked sialosides and their non PAA-linked counterparts on micro-well glass slides. This set-up, with 48 arrays on a single slide, enables simultaneous assays of 6 glycan binding proteins at 8 dilutions, interrogating 6 different glycans, including two non-sialylated controls. This is equivalent to 18x 96-well plates in the traditional plate assay. The glycan array format decreases consumption of compounds and biologicals and thus greatly enhances efficiency.

Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors.

小型化聚糖微阵列测定评定A型流感病毒血凝素的亲和力和特异性

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the ...

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant viruses may be potentially pathogenic for many species of protists. They belong to the recently described order of Megavirales. The new lineage Faustovirus that has been isolated from sewage samples is distantly related to the mammalian pathogen African swine fever virus. This virus is also specific to its amoebal host, Vermamoeba vermiformis, a protist common in health care water systems. It is crucial to continue isolating new Faustovirus genotypes in order to enlarge its genotype collection and study its pan-genome. We developed new strategies for the isolation of additional strains by improving the use of antibiotic and antifungal combinations in order to avoid bacterial and fungal contaminations of the amoeba co-culture and favoring the virus multiplication. We also implemented a new starvation medium to maintain V. vermiformis in optimal conditions for viruses co-culture. Finally, we used flow cytometry rather than microscopic observation, which is time-consuming, to detect the cytopathogenic effect. We obtained two isolates from sewage samples, proving the efficiency of this method and thus widening the collection of Faustoviruses, to better understand their environment, host specificity and genetic content.

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

We describe here the latest advances in viral isolation for the characterization of new genotypes of Faustovirus, a new asfarvirus-related lineage of giant viruses. This protocol can be applied to the high throughput isolation of viruses, especially giant viruses infecting amoeba.

一种快速战略新Faustoviruses从环境样品使用隔离<em&gt; Vermamoeba vermiformis</em

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

通过酶联凝集素测定来测量神经氨酸酶流感抑制抗体滴度

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most biological materials do not contain dense elements capable of scattering electrons to create an image; therefore, a negative stain, which places dense heavy metal salts around the sample, is required. In order to visualize viruses in suspension under the TEM they must be applied to small grids coated with a transparent surface only nanometers thick. Due to their small size and fragility, these grids are difficult to handle and easily moved by air currents. The thin surface is easily damaged, leaving the sample difficult or impossible to image. Infectious viruses must be handled in a biosafety cabinet (BSC) and some require a biocontainment laboratory environment. Staining viruses in biosafety levels (BSL)-3 and -4 is especially challenging because these environments are more turbulent and technicians are required to wear personal protective equipment (PPE), which decreases dexterity. 
In this study, we evaluated a new device to assist in negative staining viruses in biocontainment. The device is a capsule that works as a specialized pipette tip. Once grids are loaded into the capsule, the user simply aspirates reagents into the capsule to deliver the virus and stains to the encapsulated grid, thus eliminating user handling of grids. Although this technique was designed specifically for use in BSL-3 or -4 biocontainment, it can ease sample preparation in any lab environment by enabling easy negative staining of virus. This same method can also be applied to prepare negative stained TEM specimens of nanoparticles, macromolecules and similar specimens.

This protocol provides instruction for negative staining virus samples which can easily be used in BSL-2, -3, or -4 laboratories. It includes the use of an innovative processing capsule, which protects the transmission electron microscopy grid and provides the user easier handling in the more turbulent environments within biocontainment.

利用胶囊对生物达标中病毒样品的阴性染色

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.