Evaluation of Assays to Quantify Infectious Human Norovirus for Heat and High-Pressure Inactivation Studies Using Tulane Virus.

We compared the heat and high hydrostatic pressure (HHP) inactivation results of Tulane virus (TV), a human norovirus (HuNoV) surrogate, obtained by plaque assay, direct quantitative reverse transcription PCR (RT-qPCR), porcine gastric mucin magnetic beads (PGM-MBs) binding assay followed by RT-qPCR (PGM/PCR), and propidium monoazide (PMA) assay followed by RT-qPCR (PMA/PCR). Heat and HHP inactivation of a HuNoV genotype I.1 (GI.1) strain and a genotype II.4 (GII.4) strain was also evaluated using those molecular assays. Viruses were heat treated at 50-90 °C for 2 min and HHP treated at 100-550 MPa at initial temperatures of 4 or 21 °C for 2 min. For heat treatment, the three molecular methods significantly underestimated the inactivation of TV. It could be logically concluded that the PGM/PCR assay was better than the PMA/PCR and direct RT-qPCR assays in estimating the inactivation of HuNoV GI.1. The three molecular methods were comparable in estimating the heat inactivation of GII.4. For HHP treatment, both PGM/PCR and PMA/PCR assays were able to estimate inactivation of TV at ≤2-log reduction levels, but significantly underestimated its inactivation at >2-log reduction levels. The direct RT-qPCR assay was the worst method for estimating HHP inactivation of TV. It could be logically concluded that the PGM/PCR and PMA/PCR assays were comparable in estimating the HHP inactivation of GI.1 and both were significantly better than the direct RT-qPCR assay. Among the three molecular methods, the PGM/PCR assay was the best in estimating the HHP inactivation of GII.4. These results demonstrated that the PGM/PCR assay was probably the method of choice in estimating the inactivation of HuNoV GI.1 and GII.4 for heat and HHP treatments, but this method would likely result in underestimation of HuNoV inactivation.