Binding-Based RT-qPCR Assay to Assess Binding Patterns of Noroviruses to Shellfish

Outbreaks of norovirus (NoV) gastroenteritis are often associated with consumption of shellfish contaminated with human NoVs. Strong non-specific binding and specific binding between NoVs and histo-blood group antigens (HBGAs) present in shellfish tissues may explain why depuration is ineffective. Recent studies on NoV-binding patterns in shellfish have examined the attachment of NoV virus-like particles (VLPs) to HBGAs present in shellfish using enzyme-linked immunosorbent assays (ELISAs). As NoVs are genetically diverse, it is not practical to produce a range of VLPs and specific antibodies for binding studies. Tank-based bioaccumulation experiments for binding studies also require laboratory space and time. The aim of this study was to develop an alternative method to determine binding patterns for a range of shellfish species and NoV genotypes without using VLPs, antibodies, or tanks. Pacific oysters, green-lipped mussels, two GI and four GII NoV genotypes were selected for assay development. Shellfish gut homogenates were coated onto microwell plates, then purified NoV suspensions were added to each well. Blocking and wash steps using similar reagents as used in ELISAs were carried out. RNA was extracted directly in each well, then RNA copies were quantified by RT-qPCR. Diluent buffer-coated wells spiked with NoVs were used as controls. Different binding patterns were observed. NoV binding was always higher with oysters than with mussels. The highest NoV binding was found with GI.3 and oysters, with 97 % NoV GI.3 bound to oyster homogenate compared with 5 % bound to mussel homogenate. GI.4 did not bind to mussels.