Purpose: This study evaluates binding of GI.1 and GII.4 HuNoV to histo-blood group antigens expressed in porcine gastric mucins (PGM) as a surrogate for detecting infectious virus following treatment with heat or a levulinic acid plus sodium dodecyl sulfate (Lev/SDS) sanitizer.
Methods: GI.1 and GII.4 HuNoV (3-5 log genome copies/sample) were inactivated by heat (99oC for 5 min) or with liquid sanitizers containing 0.5% Lev/0.01% SDS (low SDS) or 0.5% Lev/0.1% SDS (high SDS) for 1 min. Treated and untreated (control) virus samples were applied to 96-well plates coated with 1 µg/ml PGM. RNase A (100 ng/well) was added to designated wells for each sample to degrade exposed RNA. The number of wells (positive/total) containing bound and potentially intact virus was calculated after real-time RT-PCR. Murine norovirus (MNV-1) inactivation was also assessed by plaque assay.
Results: After thermal inactivation, 10% (1/10) and 16% (1/6) of PGM coated wells were positive for GI.1 and GII.4 binding with RNase A treatment respectively whereas no binding (0/12) and 33.3% (2/6) binding was observed for non-RNase A treated samples in GI.1 and GII.4 HuNoV respectively. For both GI.1 and GII.4, the high SDS sanitizer completely eliminated PGM binding (0/18 each), but 18/18 wells treated with the low SDS sanitizer were positive. Murine norovirus infectivity correlated with PGM binding. All positive control wells were positive for GI.1 and GII.4 binding.
Significance: The PGM-binding method is a promising surrogate for discriminating between infectious and non-infectious HuNoVs after capsid destruction by high heat or high Lev/SDS sanitizer. Studying the kinetics of HuNoV inactivation and assay sensitivity is currently underway and necessary for further validation of the method.