Alternative <em>In Vitro</em> Methods for the Determination of Viral Capsid Structural Integrity

Abstract

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.

A Survey of Analytical Techniques for Noroviruses

“…Similarly, the dynamic light scattering (DLS) technique has also been investigated for NoV detection, however, high concentrations of purified capsids are required for the accurate determination of viruses by this technique [198]. The mechanism of using the DLS technique for NoV detection is that DLS can be used for detection of dispersed or aggregated viruses, as well as viral capsid proteins and dimers with the condition that the identity and purity of the analyte are known [198]. Virus aggregation is a sign of loss of capsid integrity due to the easy aggregation of disrupted capsids [198].…”

“…The mechanism of using the DLS technique for NoV detection is that DLS can be used for detection of dispersed or aggregated viruses, as well as viral capsid proteins and dimers with the condition that the identity and purity of the analyte are known [198]. Virus aggregation is a sign of loss of capsid integrity due to the easy aggregation of disrupted capsids [198].…”

Free Chlorine and Peroxynitrite Alter the Capsid Structure of Human Norovirus GII.4 and Its Capacity to Bind Histo-Blood Group Antigens

“…However, such methods cannot be used to determine the infectious risk because viral genome is more stable than viral infectivity (Gassilloud et al, 2003;Polo et al, 2018). Instead of performing direct genome quantification, some authors have suggested first evaluating HuNoV capsid integrity using the HBGA-binding feature to select infectious HuNoVs prior to genome quantification (Moore et al, 2017). To validate this approach, further basic knowledge about the alteration of the capsid protein under inactivating conditions is needed.…”