萤光素酶表达的重组病毒的高通量滴定
Summary
This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.
Abstract
Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with viruses that do not form plaques. As an alternative to plaque assays, we have developed a high-throughput titration method that allows for the simultaneous titration of a high volume of samples in a single day. This approach involves infection of the samples with a Firefly luciferase tagged virus, transfer of the infected samples onto an appropriate permissive cell line, subsequent addition of luciferin, reading of plates in order to obtain luminescence readings, and finally the conversion from luminescence to viral titers. The assessment of cytotoxicity using a metabolic viability dye can be easily incorporated in the workflow in parallel and provide valuable information in the context of a drug screen. This technique provides a reliable, high-throughput method to determine viral titers as an alternative to a standard plaque assay.
Introduction
Classical viral plaque assays continue to be a mainstay in virus research even though they can be notoriously time-consuming and constitute a significant bottleneck to obtaining results from experiments. More rapid indirect virus quantification methods have emerged including quantitative polymerase chain reaction (qPCR), ELISA, and flow cytometry1-4. Recent innovations such as the Virocyt virus counter can directly count viruses using advanced flow sorting technology and through a combination of protein and DNA/RNA dyes5. While all of these methods have undoubtedly quickened the pace of virus research, each method has its advantages and drawbacks. For example, qPCR can allow for quantification of specific viral genome sequence but cannot effectively discriminate infectious from defective virions6. ELISAs can be very specific however require a suitable antibody against the desired target viral protein and can be very expensive. While flow cytometry technology offers many advantages and has improved significantly, throughput and accessibility to highly specialized equipment nonetheless remains a hurdle. Importantly, all of these techniques are not ideally suited for high-throughput screening, for which the ease and time requirement of the virus quantification step is of critical importance.
Here we describe a high-throughput and easily automatable technique to titer viruses that express a Firefly luciferase (Fluc) transgene. This method generates approximate viral titers in a test sample based on luminescence signal reads through the parallel use of a standard curve of known amounts of virus. Samples containing unknown quantities of luciferase-expressing virus are transferred on to a permissive “plaquing” cell line in parallel with the standard virus dilution curve and virus-associated luminescence is read after a few hours incubation time. This allows for rapid, quantitative, often same-day generation of results, unlike classic plaque assay protocols which typically require several days of incubation in order to manually count visible plaques7-9.
The protocol outlines the steps of our titration method using oncolytic Vesicular Stomatitis Virus encoding a Fluc transgene (VSV∆51-Fluc) as an example and provides an overview of 1. Sample preparation 2. The plating of a permissive cell line for virus titration using an automated dispenser 3. The preparation of the viral standard curve 4. The transfer of the sample supernatants onto the permissive cell line using a 96-well manual pipettor 5. The assessment of sample cytotoxicity using a cell viability reagent 6. The preparation of the luciferin substrate 7. Reading of bioluminescence and 8. Data analysis.
Protocol
Representative Results
Discussion
这里描述的荧光素酶为基础的方法提供了许多优于其它现有的方法，包括其方便，快速，最小的设备的需要，并且成本相对较低。一个关键因素，这是避免连续稀释步骤。尽管如此，这个协议的系列稀释基衍生物是肯定可行的，最近被用于评估在高通量抗病毒药屏幕11荧光素酶表达埃博拉滴度。而本质上更耗时且较昂贵，这样的修改可以针对病毒的定量提供更大的动态范围，必要时。除了是特别适合用于评估病毒滴度在高通量筛选的上下文中，我们的一步法荧光素酶基的病毒定量方法产生在复制病毒的情况下，感染性病毒粒子的准确估计。此外，发光以及来自同一实验的细胞毒性数据读数得到更的靶细胞上的实验条件下的效果完整图像，这是在溶瘤病毒和药物屏幕的背景下尤其有用。
这里所示的例子中使用了复制的负单链RNA病毒;然而，该协议可以适应许多复制型或非复制型病毒有一些小的协议的调整。这包括DNA病毒，如牛痘，单纯疱疹病毒，和腺相关病毒（参见图5）。感染细胞内的病毒，如牛痘病毒，例如样品，需要事先向量化（ 例如 ，至少一个冷冻-解冻周期）的病毒的释放步骤。当应用该技术对其他的病毒，它是必要的，以从病毒上清液或裂解物由光度计转移到所述板的读取优化的温育时间。该参数将主要取决于病毒在允许细胞系和复制周期p的强度romoter驾驶荧光素酶的表达。这最好通过使用完整的标准曲线，在优化步骤中完成的。要做到这一点，必须有感染的编制标准曲线的各种重复的适当宽松细胞系和阅读每个重复在不同的时间点后转移。理想情况下，孵育时间点被选择，导致LOG（RLU）和LOG（效价）之间的线性关系跨越预期样品的滴度范围。为VSVΔ51-Fluc，这通常是从10 4 PFU / ml的-10 7 PFU / ml的为5小时的温育时间。如果更低或更高的滴度从样品预期，可以简单地增加或减少培养时间分别。或者，样品可以稀释到下降多达通常用于ELISA进行的范围内。
如上面所提到的，这种方法非常适合于执行使用药物库的大多数步骤可以是自动化的高通量药物屏幕。细胞可以被镀èfficiently使用自动化微板的分配器，所述药物库可使用96通道的液体处理器加入，病毒可以使用微分配器被添加和平板阅读使用自动化的光度计。从理论上讲，这也适用于384孔或更小的格式;但是，不限于该端是多个小区可以被镀的，给予较少的细胞导致更窄的范围中的LOG（RLU）的线性度，以LOG（效价）的关系。最后，使用刃天青或其他代谢染料细胞活力评估可以很容易地在工作流中引入，从而允许细胞毒性化合物的抗病毒帘或鉴定的化合物，导致协同杀伤与病毒12组合判断。然而，这种方法的局限性包括：荧光素酶转基因表达的病毒，这并不总是可能的要求，并具有足够允许细胞系的可用性。然而，这很可能能够适应该方法与其它报告基因的使用（ 例如 ，GFP）提供记者定量方法具有适当的线性度和信噪比。总体而言，所描述的高通量的方法可以进行修改，以适应许多不同的病毒，并适用于多种应用。
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
Vanessa Garcia is funded by a Queen Elizabeth II Ontario Graduate Scholarship in Science and Technology and Cory Batenchuk by a Natural Sciences and Engineering Research Council fellowship.
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| Dulbeccos' Modified Eagle's Medium (DMEM) | Corning | SH30243.01 | |
| Fetal Bovine Serum | NorthBio Inc. | NBSF-701 | |
| Phosphate buffered saline | Corning | 21-040-CV | |
| HEPES | Fisher Scientific | BP310-1 | Prepare a 1mM solution, pH 7.3 |
| alamarBlue | AbD Serotec | BUF012B | |
| D-Luciferin potassium salt | Biotium | 10101-2 | |
| 96-well solid white flat bottom polystyrene TC-treated microplates | Corning | 3917 | 384-well plates can also be used for higher throughput |
| Synergy Mx | BioTek | SMTBL | Monochromator microplate reader |
| Liquidator96 | Mettler Toledo | LIQ-96-200 | 96 tip manual pipetting system |
| Liquidator96 LTS Tips sterilized with filters | Mettler Toledo | LQR-200F | Any sterile filtered tips compatible with pipettors of choice are appropriate |
| Microflo | BioTek | 111-206-21 | Used to plate cells in a 96 well plate |
| Fluoroskan Ascent FL | Thermo Scientific | 5210450 | Microplate fluorometer |
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