Moore Matthew D, Mertens Brittany S, Jaykus Lee-Ann
Department of Food, Bioprocessing, and Nutrition Sciences, North Carolina State University;
Department of Chemical and Biomolecular Engineering, North Carolina State University.
J Vis Exp. 2017 Nov 16(129):56444. doi: 10.3791/56444.
Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine detection of the virus. The current detection and quantification techniques, which rely primarily on nucleic acid amplification, do not discriminate infectious from non-infectious viral particles. The purpose of this article is to present specific details on recent advances in techniques used together in order to acquire further information on the infectivity status of viral particles. One technique involves assessing binding of a norovirus ssDNA aptamer to capsids. Aptamers have the advantage of being easily synthesized and modified, and are inexpensive and stable. Another technique, dynamic light scattering (DLS), has the advantage of observing capsid behavior in solution. Electron microscopy allows for visualization of the structural integrity of the viral capsids. Although promising, there are some drawbacks to each technique, such as non-specific aptamer binding to positively-charged molecules from sample matrices, requirement of purified capsid for DLS, and poor sensitivity for electron microscopy. Nonetheless, when these techniques are used in combination, the body of data produced provides more comprehensive information on norovirus capsid integrity that can be used to infer infectivity, information which is essential for accurate evaluation of inactivation methods or interpretation of virus detection. This article provides protocols for using these methods to discriminate infectious human norovirus particles.
人诺如病毒在全球范围内造成了相当大的公共卫生和经济损失。新兴的体外培养技术进展尚未适用于该病毒的常规检测。当前的检测和定量技术主要依赖核酸扩增，无法区分感染性和非感染性病毒颗粒。本文的目的是详细介绍共同使用的技术的最新进展，以便获取有关病毒颗粒感染状态的更多信息。一种技术涉及评估诺如病毒单链DNA适配体与衣壳的结合。适配体具有易于合成和修饰、价格低廉且稳定的优点。另一种技术，动态光散射（DLS），具有观察衣壳在溶液中行为的优势。电子显微镜可以可视化病毒衣壳的结构完整性。尽管很有前景，但每种技术都有一些缺点，例如适配体与样品基质中的带正电分子的非特异性结合、DLS需要纯化的衣壳以及电子显微镜的灵敏度较低。尽管如此，当这些技术结合使用时，所产生的数据提供了关于诺如病毒衣壳完整性的更全面信息，可用于推断感染性，这些信息对于准确评估灭活方法或解释病毒检测至关重要。本文提供了使用这些方法区分感染性人诺如病毒颗粒的方案。
