Model for Estimating Norovirus Infectivity

March 2014

Human noroviruses (HuNoV) are a major source of foodborne illnesses worldwide. As they cannot be cultured in vitro, alternative methods that discriminate between infectious and non-infectious HuNoV are needed. A promising method for detecting infectious HuNoV by binding to porcine gastric mucins (PGM) and treatment with RNase A, followed by RT-PCR was recently developed. HuNoVs specifically recognize histo-blood group antigens (HBGA) expressed on the human gastrointestinal mucosa and also on PGM. Binding to HBGAs is a necessary precursor to human infection for some HuNoV strains. These strains must have an intact (not damaged) HBGA-binding site on their viral capsid in order to bind to HBGAs and they must have an intact viral RNA genome in order to cause infection in a host cell.

Treating viruses with RNase A is another means of discriminating between intact and non-intact viruses, as the genomes of intact viruses are protected from the RNA-degrading enzyme. Therefore, HuNoVs are considered to be potentially infectious when they 1) bind to HBGAs found in PGM, 2) are resistant to RNase A treatment, and 3) can be subsequently detected by RT-PCR.

Genogroup I genotype 1 (GI.1) and Genogroup II, genotype 4 (GII.4) HuNoV were inactivated with heat or a levulinic acid plus sodium dodecyl sulfate (Lev/SDS) sanitizer. The PGM-binding method described above was evaluated as a surrogate for discriminating between infectious and non-infectious HuNoV in the inactivated samples.

GI.1 and GII.4 HuNoV (3-5 log genome copies/100-µl sample) were inactivated by heat treatment (99°C 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) HuNoV were deposited in high-binding, 96-well plates coated with 1 µg/ml PGM suspended in 50 mM carbonate-bicarbonate solution (CBS). RNase A (100 ng) was added to each well to degrade the exposed RNA.

With each of these experimental parameters (PGM-binding and RNase A degradation), control samples were included; wells coated with CBS alone and a set of samples not treated with RNase A. The number of wells containing bound and potentially intact virus was calculated after real-time RT-PCR analysis of RNA extracted from each well. Using the same inactivation treatments, Murine norovirus (MNV-1), a cultivable norovirus surrogate, was also assessed by plaque assay.

After thermal inactivation, 10% (1/10) and 16.7% (1/6) of PGM-coated wells treated with RNase A were positive for GI.1 and GII.4 binding, respectively, whereas no binding (0/12) and 33.3% (2/6) binding, respectively, were observed for in GI.1 and GII.4 HuNoV not treated with RNase A. For both GI.1 and GII.4, treatment with the high SDS sanitizer eliminated binding in 18 wells, but 18/18 wells treated with 0.5% Lev/0.01% SDS sanitizer were positive for virus PGM-binding.

Murine norovirus infectivity was eliminated by heat and sanitizer treatments using the two treatment methods, as determined by plaque assay. This observation correlated with PGM binding for HuNoV inactivated with Lev/SDS sanitizer, but not completely with heat inactivated HuNoV. PGM binding was observed in heat inactivated GII.4 samples. All positive control (untreated) wells were positive for GI.1 and GII.4 binding throughout the study, but non-specific binding in uncoated wells was also apparent for untreated HuNoV.

The PGM-binding method is a promising surrogate for discriminating between infectious and non-infectious HuNoVs after capsid destruction by heating at 99ºC or treatment with a 0.5% Lev/0.1% SDS sanitizer. Studies on the kinetics of HuNoV inactivation and the assay sensitivity and specificity are needed for further validation of the method.