Elucidating Human Norovirus Attachment to the Surface of Strawberries

Introduction: Fresh berries, including raspberries and strawberries, have been associated with human norovirus outbreaks. Although norovirus has been shown to bind to berries, the specific berry components that contribute to virus attachment are unknown. Candidate mechanisms for attachment include histo-blood group-like antigens (HBGAs) or other unknown ligands in berry tissue; native berry microflora; and/or electrostatic and hydrophobic interactions between fruit and virus.

Purpose: To identify strawberry characteristics and/or components which contribute to human norovirus attachment to berries. 

Methods: Two cultivars of whole strawberries and berry pistils were collected. The binding affinity of human norovirus GI.6 and GII.4 (Sydney) to berry surface slices and pistils was determined by RT-qPCR-based suspension assays. Visual localization of GII.4 Virus-Like Particles (VLPs) on berry surfaces was performed by confocal microscopy, and VLP adherence to pistils was confirmed by transmission electron microscopy (TEM). To determine the specificity of norovirus interactions to pistils, zeta potential, carbohydrate composition, and carbohydrate linkage analyses were performed. Additionally, a lectin-binding competitive ELISA was used to determine if specific strawberry sugar moieties contributed to norovirus binding.

Results: Based on suspension assay results, >90% and >94% (P< 0.05) of input norovirus bound to strawberry surfaces and pistils, respectively. Fluorescently labeled VLPs on the strawberry surface were primarily located on broken pistils and achenes. TEM results showed VLP attachment on pistils, with aggregation occurring on disrupted pistil surfaces. Physiochemical analyses of pistils showed a highly electronegative zeta potential (pH range 3.5 to 9). Glucose, arabinose, and galactose were the primary terminal sugars on complex carbohydrates of strawberries. Exposing purified strawberry carbohydrates to lectins significantly reduced VLP binding affinity.  

Significance: Human norovirus binds tenaciously to strawberry surfaces and pistils, particularly regions of injury. Results suggest that specific carbohydrates facilitate virus binding to strawberries and efforts to identify these ligands continue.