Name: zika virus specific diagnostic epitope discovery
Uploaded: 2017-12-12T13:00:00.000+00:00
Duration: 11 min 37 s
Description: Watch this Scientific Journal Video about Zika Virus Specific Diagnostic Epitope Discovery at JoVE.com

目前的支持由 NIDCRR44 DE024456 的 SBIR (小型企业创新研究) 行政补充补助金提供。NIDCR 艾滋病毒补助金的发展 NIDCR 赠款 U01 DE017855, 以制定一个验证性的护理点诊断艾滋病毒。我们感谢 Silke Weisenburger (德国海德堡 PEPperPRINT) 的技术援助和亲切支持。我们还感谢纽约大学朗格尼医学中心的李-林奥德赛成像系统。

这些数据是纽约大学牙科学院正在进行的研究研究的一部分, 由纽约大学医学院的机构审查委员会批准, H10-01894。本研究中使用的临床样本是以前用于诊断的不确定样本, 并得到纽约州奥尔巴尼的美国卫生部的沃斯德中心的许可。
1. 在特定的孵化盘中安装 ZIKV 高密度肽微阵列幻灯片
<ol><li>使用无粉手套处理玻璃滑边。玻璃滑动尺寸是75.4 毫米25.0 毫米和1毫米厚。将幻灯片放在孵化盘中, 微阵列表面朝上。</li>
	<li>将密封面的光泽面向下放置在微阵列打印表面上。为了保证良好的位置, 请将密封件和底板的螺钉孔重叠。</li>
	<li>将托盘上部放在密封上, 以创建微阵列腔。</li>
	<li>固定在托盘中的幻灯片, 通过在一个交替的模式, 从左上角开始, 然后右下角, 同样地拧紧螺钉。放置孵化盘的盖子。</li>
</ol>2. 背景相互作用检测: 用二次抗体染色微阵列
注: 使用吸管将溶液 (标准和阻塞缓冲器, 稀释后的二级抗体) 存入芯片室的拐角处。
<ol><li>用标准缓冲器 (1x 磷酸盐缓冲盐水 (PBS), 0.05% 吐温 20, pH 值 7.4, 用0.45 µm 过滤器 (总容积2000µL/微阵列) 孵化芯片, 在 rpm 的轨道振动筛室温下 (RT) 进行15分钟的检测。</li>
	<li>通过从微阵列腔的拐角处吸出吸管来去除标准缓冲器。用空白幻灯片填充空幻灯片持有者, 以防止微阵列幻灯片的中断。</li>
	<li>在 140 rpm 的轨道振动筛上, 用阻塞缓冲器 (总容积2000µL/微阵列) 拦截60分钟的微阵列。</li>
	<li>在微阵列腔的拐角处用吸管抽吸, 以去除堵塞缓冲器。</li>
	<li>用二级抗体孵化微阵列, 抗人 IgM fluorochrome 共轭, 稀释 1: 5000 (总容积2000µL/微阵列) 在染色缓冲器 (10% 阻塞缓冲器在标准缓冲器) 为30分钟在黑暗中 RT 在一个轨道振动筛在140rpm.</li>
	<li>在微阵列腔的拐角处用吸管吸除二次抗体。</li>
	<li>用标准缓冲器清洗微阵列 3x, 每洗1分钟 (总容积为2000µL/微阵列, 在 140 rpm 的轨道振动筛上。每次清洗后, 通过从芯片室的拐角处吸出吸管来去除标准缓冲器。</li>
	<li>将幻灯片2x 浸入新鲜准备的浸渍缓冲 (1 毫米, pH 值 7.4), (总体积为200毫升)。</li>
	<li>仔细地干燥微阵列, 大约1分钟, 通过从顶部到幻灯片底部非常仔细地吸出多余的液体而不触及滑动面。分析微阵列扫描仪读取器中的幻灯片。</li>
</ol>3. 微阵列暴露于宿主血清中
注意: 在实验室安全条件下执行这一步骤, 生物安全等级 2 (BSL-2), 因为血清标本的潜在感染性。在第二类生物安全柜 (BSC) 内工作。
<ol><li>在4°c13的5分钟内, 在1.6万个区域合作框架内对30分钟和离心机进行56摄氏度灭活血清样品。 注: 使用吸管将溶液 (染色, 标准缓冲液和稀释后的血清) 存入芯片室的角落。</li>
	<li>稀释血清样品在染色缓冲从 1:1, 000 (总容量 2000 ul/微阵列) 稀释。保持在4摄氏度, 直到使用。</li>
	<li>用染色缓冲器 (2000 ul/微阵列的总容积) 孵化微阵列, 在 140 rpm 的轨道振动筛上进行15分钟的 RT。</li>
	<li>在微阵列腔的拐角处用吸管吸出, 去除染色缓冲器。</li>
	<li>用稀释后的血清样本在 140 rpm 的轨道振动筛上过夜, 将微阵列孵化为4摄氏度。</li>
	<li>在微阵列的拐角处用吸管吸出稀释后的血清样品。</li>
	<li>用标准缓冲器清洗微阵列 3x, 每洗1分钟 (总容积 2000 ul/微阵列在轨道振动筛 140 rpm。每次清洗后, 通过从芯片室的拐角处吸出吸管来去除标准缓冲器。</li>
</ol>4. 用二次抗体染色和标记控制抗体
注: 使用吸管将溶液 (标准缓冲器, 稀释的二次和标签抗体) 存入芯片室的拐角处。
<ol><li>稀释染色缓冲器中的二次抗体。 注: 使用抗人 IgM fluorochrome 共轭抗体或抗人 IgG fluorochrome 共轭抗体稀释 1:5, 000 (总容积2000µL/微阵列) 染色缓冲器。</li>
	<li>混合控制标签抗体 (单克隆抗 HA fluorochrome 共轭) 在稀释 1:1, 000 (总容量 2 000 µL/微阵列) 在染色缓冲器与二次抗体以前稀释在染色缓冲。</li>
	<li>用微阵列孵育30分钟, 在黑暗的轨道振动筛在 140 rpm。</li>
	<li>在微阵列的拐角处用吸管抽吸, 去除稀释后的二级和标签抗体的混合。</li>
	<li>用标准缓冲器清洗 3x (总容积为2000µL/微阵列)。每个洗涤是1分钟在一个轨道振动筛在140转每分钟。每次清洗后, 通过从芯片室的拐角处吸出吸管来去除标准缓冲器。</li>
</ol>5. 微阵列扫描
<ol><li>将幻灯片2x 浸入新鲜准备的浸渍缓冲液 (1 毫米, pH 值 7.4) (总体积为200毫升)。</li>
	<li>仔细地干燥芯片, 大约1分钟, 通过非常小心地从顶部到幻灯片底部, 而不触及幻灯片表面。</li>
	<li>按照扫描仪的说明扫描微阵列。将幻灯片置于扫描仪上, 打印面朝上。</li>
	<li>创建一个新项目并选择扫描区域。将扫描仪参数设置为: 分辨率:21 µm, 强度为700和 800 nm 通道: 7.0, 扫描质量: 中和偏移: 0.8。</li>
	<li>获取并保存扫描原始图像作为16位灰度 TIFF 文件格式。</li>
</ol>6. 使用特定软件的微阵列分析
<ol><li>打开原始图像 (TIFF 图像)。打开阵列网格文件。</li>
	<li>使用计算机的鼠标或键盘箭头键将阵列网格与扫描图像对齐。</li>
	<li>在特定软件中选择 "量化选择", 它创建一个读数文件, 其中包含每个点的信号强度、背景值和相应的肽序列。 注意: 此输出文件还可以导入到电子表格程序中, 以进行其他分析和绘图。</li>
</ol>7. 微阵列存储
<ol><li>储存在黑暗中密封的微阵列在4°c 下无氧氮气或氩气。</li>
</ol>

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作者没有什么可透露的。

我们设计了一个协议, 利用高密度肽芯片包含整个 Zika 病毒蛋白序列 (法国玻利尼西亚人菌株)。该芯片是通过打印3423不同的重叠线性多肽制造的。每一个肽是15氨基酸并且由它的最近的邻居仅一个残余在序列 (例如, 14 残余重叠) 变化。虽然可以打印较短的重叠, 但表位映射更精确, 重叠时间更长。每一个肽都以重复打印, 以提高可靠性。
重要的是开始的过程中, 预先染色?...

基于临床症状的 Zika 病毒 (ZIKV) 诊断具有挑战性, 因为它与登革热和基孔肯亚病毒感染的传播媒介、地理分布和症状有关1。鉴于怀孕期间感染 ZIKV 的妇女面临不良妊娠结局的风险, 区分3病毒是很重要的。虽然目前的分子诊断测试是特定的, 但它们只在血液或唾液中有用, 在相对较短的急性感染期间2,3。血清学检测是诊断这一初始感染期以外的重要4。
ZIKV 特异性血清学检测的发展具有挑战性的两个原因: 一是人类免疫系统反应的 Zika 抗原目前尚不为人所知;第二, 保守的 flaviviruses 氨基酸序列诱导抗体的交叉反应性。我们的目的是发现独特的 ZIKV 特定肽用于诊断。已经开发出不同的方法来筛选包括噬菌体、细菌和酵母表面在内的整个蛋白质的肽库, 显示5,6,7,8,9, 10。我们的战略是使用高密度肽芯片, 允许快速和廉价的高通量血清学筛查11,12 , 随后确定的肽可用于改善目前的血清学检测 ZIKV 感染的方法。
此协议允许使用高密度肽芯片 (图 1) 发现 Zika 病毒特定诊断肽。采用多肽激光印花技术制备高密度肽微阵列。整个 ZIKV 蛋白序列由3423种基于法国玻利尼西亚应变 (基因库: KJ776791.2) 的氨基酸残留物印在标准玻璃上, 在15条线性残留物块中, 重叠14个氨基酸残留物, 以复制共6846个肽斑。除了整个 Zika 蛋白序列多肽, 微阵列利用流感血凝素 (HA) 肽进行内部控制. 
Zika 证实的阳性血清样本 (纽约州奥尔巴尼) 获得, 用于鉴定特定的免疫球蛋白 M (IgM) 和免疫球蛋白 G (IgG) 活性肽。在一夜之间用样本孵化后, 微阵列被染成二级 fluorochrome 共轭抗体 (抗人 IgM 或抗人 IgG), 并在微阵列扫描仪上进行分析。用制造芯片的同一公司提供的特定软件对光斑强度和肽标注进行量化。

<table><tbody><tr><td>PEPperCHIP Custom Peptide Microarray</td><td>PEPperPRINT</td><td>PPC.001.001</td><td>Custom peptide micarray: our microarray contains the entire Zika virus protein sequence</td></tr><tr><td>PEPperCHIP incubation tray 3/1</td><td>PEPperPRINT</td><td>PPC.004.001</td><td></td></tr><tr><td>PEPperCHIP Staining Kit (680 nm) (anti-HA, DyLight labeling)</td><td>PEPperPRINT</td><td>PPC.037.002</td><td></td></tr><tr><td>PepSLide Analyzer</td><td>PEPperPRINT</td><td>PSA.004.001</td><td>14-days free License for Windows</td></tr><tr><td>Rockland Blocking buffer</td><td>Rockland</td><td>MB-070</td><td></td></tr><tr><td>anti-human IgM (mu chain) DyLight 800</td><td>Rockland</td><td>609-145-007</td><td></td></tr><tr><td>anti-human IgG Fc DyLight 680</td><td>Thermo Scientific</td><td>SA5-10138</td><td></td></tr><tr><td>LI-COR Odyssey Imaging System</td><td>LI-COR</td><td></td><td></td></tr><tr><td>Orbital shaker device</td><td>IKA</td><td>MTS 2/4 digital microtiter shaker</td><td></td></tr><tr><td>Adobe Illustrator</td><td>Adobe</td><td></td><td></td></tr><tr><td>Deltagraph</td><td>Redrocks</td><td></td><td></td></tr></tbody></table>

zika virus specific diagnostic epitope discovery

高密度肽微阵列允许在一张标准显微镜幻灯片上筛选超过6000多肽。该方法可应用于药物的发现、治疗目标的识别和诊断的发展。在这里, 我们提出了一个协议, 以发现特定的 Zika 病毒 (ZIKV) 诊断肽使用高密度肽芯片。用一种高密度肽微阵列对 ZIKV 感染进行了验证, 该芯片含有将整个 ZIKV 蛋白转化为3423个独特的15线性氨基酸 (aa) 残留物, 并重复打印 14 aa 的残留重叠。在同一阵列内染色不同的二次抗体, 我们检测到与血清中存在的免疫球蛋白 M (IgM) 和免疫球蛋白 G (IgG) 抗体结合的肽。选择这些肽进行进一步的验证实验。在本协议中, 我们描述了在设计、处理和分析高密度肽芯片时所采用的策略。

由制造芯片的同一公司提供的特定软件对扫描后的微阵列进行了分析。所创建的读出文件包含每个点的信号强度、背景值和相应的肽序列。通过绘制在从原始值中减去背景后获得的绿色前景中值 (图 3), 可以直观地描绘出每个肽对 IgM 有强烈反应的斑点荧光强度。
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Watch this Scientific Journal Video about Zika Virus Specific Diagnostic Epitope Discovery at JoVE.com

Zika Virus Specific Diagnostic Epitope Discovery

在本协议中, 我们描述了一种利用高密度肽芯片来发现 Zika 病毒特异性诊断肽的技术。该议定书可 readly 其他新出现的传染性疾病。

Zika 病毒特定诊断表位发现

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for ...

zika-virus-specific-diagnostic-epitope-discovery

Research

JoVE Journal

Immunology and Infection

7.7K 次观看.  New York University College of Dentistry. 在本协议中, 我们描述了一种利用高密度肽芯片来发现 Zika 病毒特异性诊断肽的技术。该议定书可 readly 其他新出现的传染性疾病。

视频: Zika Virus Specific Diagnostic Epitope Discovery

Multiplexed Isothermal Amplification Based Diagnostic Platform to Detect Zika, Chikungunya, and Dengue 1

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different downstream patient-to-patient transmission potentials, and require very different patient treatments. Thus, recent Zika outbreaks make it urgent to develop tools that rapidly discriminate these viruses in patients and trapped mosquitoes, to select the correct patient treatment, and to understand and manage their epidemiology in real time.
Unfortunately, current diagnostic tests, including those receiving 2016 emergency use authorizations and fast-track status, detect viral RNA by reverse transcription polymerase chain reaction (RT-PCR), which requires instrumentation, trained users, and considerable sample preparation. Thus, they must be sent to &#34;approved&#34; reference laboratories, requiring time. Indeed, in August 2016, the Center for Disease Control (CDC) was asking pregnant women who had been bitten by a mosquito and developed a Zika-indicating rash to wait an unacceptable 2 to 4 weeks before learning whether they were infected. We very much need tests that can be done on site, with few resources, and by trained but not necessarily licensed personnel.
This video demonstrates an assay that meets these specifications, working with urine or serum (for patients) or crushed mosquito carcasses (for environmental surveillance), all without much sample preparation. Mosquito carcasses are captured on paper carrying quaternary ammonium groups (Q-paper) followed by ammonia treatment to manage biohazards. These are then directly, without RNA isolation, put into assay tubes containing freeze-dried reagents that need no chain of refrigeration. A modified form of reverse transcription loop-mediated isothermal amplification with target-specific fluorescently tagged displaceable probes produces readout, in 30 min, as a three-color fluorescence signal. This is visualized with a handheld, battery-powered device with an orange filter. Forward contamination is prevented with sealed tubes, and the use of thermolabile uracil DNA glycosylase (UDG) in the presence of dUTP in the amplification mixture.

Current multiplexed diagnostics to detect Zika, chikungunya, and dengue viruses require complex sample preparation and expensive instrumentation, and are difficult to use in low resource environments. We show a diagnostic that uses isothermal amplification with target-specific strand displaceable probes to detect and differentiate these viruses with high sensitivity and specificity.

基于复用等温放大诊断平台检测 Zika、基孔肯亚和登革热1

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different ...

科研

生物化学

Reverse Transcription-Loop-mediated Isothermal Amplification (RT-LAMP) Assay for Zika Virus and Housekeeping Genes in Urine, Serum, and Mosquito Samples

Infection with Zika virus (ZIKV) can be asymptomatic in adults, however, infection during pregnancy can lead to miscarriage and severe neurological birth defects. The goal of this protocol is to quickly detect ZIKV in both human and mosquito samples. The current gold standard for ZIKV detection is quantitative reverse transcription PCR (qRT-PCR); reverse transcription loop-mediated isothermal amplification (RT-LAMP) may allow for a more efficient and low-cost testing without the need for expensive equipment. In this study, RT-LAMP is used for ZIKV detection in various biological samples within 30 min, without first isolating the RNA from the sample. This technique is demonstrated using ZIKV infected patient urine and serum, and infected mosquito samples. 18S ribosomal ribonucleic acid and actin are used as controls in human and mosquito samples, respectively.

Reverse Transcription-Loop-mediated Isothermal Amplification RT-LAMP Assay for Zika Virus and Housekeeping Genes in Urine, Serum, and Mosquito Samples

This protocol provides an efficient and low-cost method to detect Zika virus or control targets in human urine and serum samples or in mosquitoes by reverse transcription loop-mediated isothermal amplification (RT-LAMP). This method does not require RNA isolation and can be done within 30 min.

反向转录-环介导的等温放大 (RT 灯) 测定尿液、血清和蚊子标本中的 Zika 病毒和管家基因

Infection with Zika virus (ZIKV) can be asymptomatic in adults, however, infection during pregnancy can lead to miscarriage and severe neurological birth ...

遗传学

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV is an RNA virus of the Flaviviridae family. ZIKV is mainly transmitted by mosquitoes, but can also be spread by maternal to fetal vertical transmission as well as sexual contact. To date, there are no reliable treatment or vaccine options available to protect those infected by the virus. The development of a reproducible, effective Zika virus infectious cell culture system is critical for studying the molecular mechanisms of ZIKV replication as well as drug and vaccine development. In this regard, a protocol describing a mammalian cell-based in vitro Zika virus culture system for viral production and growth analysis is reported here. Details on the formation of plaques by Zika virus on a cell monolayer and plaque assay for measuring viral titer are presented. Viral genome replication kinetics and double-stranded RNA genome replicatory intermediates are determined. This culture platform was utilized to screen against a library of a small set of cytokines resulting in the identification of interferon-&#945; (IFN-&#945;), IFN-&#946; and IFN-&#947; as potent inhibitors of Zika viral growth. In summary, an in vitro infectious Zika viral culture system and various virological assays are demonstrated in this study, which has the potential to greatly benefit the research community in elucidating further the mechanisms of viral pathogenesis and the evolution of viral virulence. Antiviral IFN-alpha can further be evaluated as a prophylactic, post-exposure prophylactic, and treatment option for Zika virus infections in high-risk populations, including infected pregnant women.

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV), an emerging pathogen, is linked to fetal developmental abnormalities and microcephaly. The establishment of an effective infectious cell culture system is crucial for studies of ZIKV replication as well as vaccine and drug development. In this study, various virological assays pertaining to ZIKV are illustrated and discussed.

兹卡病毒感染细胞培养系统和<em&gt;体外</em&gt;干扰素的预防作用

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV ...

免疫与感染

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 ...

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity of antibodies or serum to enhance infection in impermissible cell lines have relied on using viral output in the media followed by plaque assays to quantify infection. More recently, these assays have examined Dengue virus (DENV) infection in the cell lines using fluorescently labeled antibodies. Both these approaches have limitations that restrict the widespread use of these techniques. Here, we describe a simple in vitro assay using Dengue virus reporter viral particles (RVPs) that express green fluorescent protein and K562 cells to examine antibody dependent enhancement (ADE) of DENV infection using serum that was obtained from rhesus macaques 16 weeks after infection with Zika virus (ZIKV). This technique is reliable, involves minimal manipulation of cells, does not involve the use of live replication competent virus, and can be performed in a high throughput format to get a quantitative readout using flow cytometry. Additionally, this assay can be easily adapted to examine antibody dependent enhancement (ADE) of other flavivirus infections such as Yellow Fever virus (YFV), Japanese Equine Encephalitis virus (JEEV), West Nile virus (WNV) etc. where RVPs are available. The ease of setting up the assay, analyzing the data, and interpreting results makes it highly amenable to most laboratory settings.

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

We describe a simple and easy protocol for measuring antibody dependent enhancement of infection by Zika virus convalescent serum using Dengue virus Reporter Viral Particles.

基于流式细胞仪的 Zika 病毒疗养血清测定登革热病毒的体外抗体依赖性

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity ...

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

感染 Ifnar1 小鼠 Immunoprivileged 器官寨卡病毒特异 T 细胞反应的评价

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...

Quantification of Antibody-dependent Enhancement of the Zika Virus in Primary Human Cells

The recent emergence of the flavivirus Zika and neurological complications, such as Guillain-Barr&#233; syndrome and microcephaly in infants, has brought serious public safety concerns. Among the risk factors, antibody-dependent enhancement (ADE) poses the most significant threat, as the recent re-emergence of the Zika virus (ZIKV) is primarily in areas where the population has been exposed and is in a state of pre-immunity to other closely related flaviviruses, especially dengue virus (DENV). Here, we describe a protocol for quantifying the effect of human serum antibodies against DENV on ZIKV infection in primary human cells or cell lines.

We describe a method to evaluate the effect of pre-existing immunity against dengue virus on the Zika virus infection by using human serum, primary human cells, and infection quantification by quantitative real-time polymerase chain reaction.

原代人细胞中寨卡病毒抗体依赖性增强的定量研究

The recent emergence of the flavivirus Zika and neurological complications, such as Guillain-Barr&#233; syndrome and microcephaly in infants, has brought ...

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

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

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

从细菌人工染色体cDNA克隆中拯救重组寨卡病毒

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

Isolation and Quantification of Zika Virus from Multiple Organs in a Mouse

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral load. The purpose of the procedure is to quantify viral titers in peripheral and CNS areas of the mouse at different time points post infection or under different experimental conditions to identify virologic and immunological factors that regulate Zika virus infection. The organ isolation procedures demonstrated allow for both focus forming assay quantification and quantitative PCR assessment of viral titers. The rapid organ isolation techniques are designed for the preservation of virus titer. Viral titer quantification by focus forming assay allows for the rapid throughput assessment of Zika virus. The benefit of the focus forming assay is the assessment of infectious virus, the limitation of this assay is the potential for organ toxicity reducing the limit of detection. Viral titer assessment is combined with quantitative PCR, and using a recombinant RNA copy control viral genome copy number within the organ is assessed with low limit of detection. Overall these techniques provide an accurate rapid high throughput method for the analysis of Zika viral titers in the periphery and CNS of Zika virus infected animals and can be applied to the assessment of viral titers in the organs of animals infected with most pathogens, including Dengue virus.

The goal of the protocol is to demonstrate the techniques used to investigate viral disease by isolating and quantifying Zika virus, from multiple organs in a mouse following infection.

从小鼠多个器官分离和定量寨卡病毒

The methods being presented demonstrate laboratory procedures for the isolation of organs from Zika virus infected animals and the quantification of viral ...

High-throughput Antiviral Assays to Screen for Inhibitors of Zika Virus Replication

Antiviral drug discovery requires the development of reliable biochemical and cellular assays that can be performed in high-throughput screening (HTS) formats. The flavivirus non-structural (NS) proteins are thought to co-translationally assemble on the endoplasmic reticulum (ER) membranes, forming the replication complex (RC). The NS3 and NS5 are the most studied enzymes of the RC and constitute the main targets for drug development due to their crucial roles in viral genome replication. NS3 protease domain, which requires NS2B as its cofactor, is responsible for the cleavage of the immature viral polyprotein into the mature NS proteins, whereas NS5 RdRp domain is responsible for the RNA replication. Herein, we describe in detail the protocols used in replicon-based screenings and enzymatic assays to test large compound libraries for inhibitors of the Zika virus (ZIKV) replication. Replicons are self-replicating subgenomic systems expressed in mammalian cells, in which the viral structural genes are replaced by a reporter gene. The inhibitory effects of compounds on viral RNA replication can be easily evaluated by measuring the reduction in the reporter protein activity. The replicon-based screenings were performed using a BHK-21 ZIKV replicon cell line expressing Renilla luciferase as a reporter gene. To characterize the specific targets of identified compounds, we established in-vitro fluorescence-based assays for recombinantly expressed NS3 protease and NS5 RdRp. The proteolytic activity of the viral protease was measured by using the fluorogenic peptide substrate Bz-nKRR-AMC, while the NS5 RdRp elongation activity was directly detected by the increase of the fluorescent signal of SYBR Green I during RNA elongation, using the synthetic biotinylated self-priming template 3&#8242;UTR-U30 (5&#39;-biotin-U30-ACUGGAGAUCGAUCUCCAGU-3&#39;).

In this work, we describe the protocols used in replicon-based and viral enzyme-based assays to screen for inhibitors of Zika virus replication in a high-throughput screening format.

高通量抗病毒检测筛查寨卡病毒复制抑制剂

Antiviral drug discovery requires the development of reliable biochemical and cellular assays that can be performed in high-throughput screening (HTS) ...