Norovirus Binding to Berries May be Independent of the Presence of HBGA-like Moieties and is Potentially Related to Interactions with Natural Microflora

Introduction: Fresh produce, including berries and leafy greens, are commonly associated with human norovirus (HuNoV) outbreaks.  HuNoVs bind to histo-blood group-like antigens (HBGAs) in foods.  Little is known about the dynamics of HuNoV binding to berries, which may be ligand (HBGA) mediated and/or facilitated by the native microflora of the berry.

Purpose: To characterize HuNoV binding to berries. 

Methods: Three varietals of strawberries and five of raspberries were collected and concentrates prepared.  The strength of HuNoV GI.1 and GII.4 virus-like particle (VLP) binding to berry concentrates was determined by an ELISA, as was the presence of HBGA-like moieties (A, B, H, Le^a, Le^y).  To isolate the natural microflora, whole berries were rinsed in PBS, the eluent plated on selective and non-selective media, and whole colony sequencing done on randomly selected colonies.  Biofilm production of selected strains was quantified using a crystal violet biofilm assay.  

Results: ELISA results indicated moderate binding of GI.1 and GII.4 VLPs to both berry concentrates (+/- absorbance ratio of 2.0-4.0).  Very low levels of Le^a HBGA-like moieties were present in strawberries (P < 0.05), and A, B, H and Le^y moieties were not detected in either berry type.  A total of 172 (n = 98 from raspberries; n = 74 from strawberries) bacterial isolates were sequenced, with the microbial profiles differing by berry type. Biofilm production also varied greatly, but strains of Pantoea agglomerans, Pseudomonas moraviensis, Klebsiella oxytoca, and Enterobacteriaceae spp. were the strongest biofilm producers (A₄₀₅> 2.2).  Ongoing studies show that VLPs bind to extracted extracellular polymeric substances (EPS) from some of these strains (+/- absorbance ratio > 2.0).  

Significance: HuNoV VLPs bind to berries that lack HBGA-like moieties and potentially to components of the natural microflora of berries. By identifying structural elements that bind viruses, targeted HuNoV removal and inactivation strategies can be developed for berries.