Viability RT-qPCR improved estimation of viral inactivation at extreme thermal and high pressure exposures over direct RT-qPCR, but human intestinal enteroids provided a more robust model.
Does viability RT-qPCR accurately predict human norovirus infectivity compared to replication in human intestinal enteroids?
While viability RT-qPCR improves estimation of norovirus inactivation under extreme conditions, human intestinal enteroids remain a more robust model for assessing true infectivity.
Viability RT-qPCR, a molecular detection method combining viability marker pre-treatment with RT-qPCR, has been proposed to infer infectivity of viruses which is particularly relevant for non-culturable viruses or sophisticated cell culture systems. Being human noroviruses (HuNoV) most frequently associated with foodborne outbreaks, this study compared different viability techniques and infectivity in human intestinal enteroids (HIE) to ultimately determine whether the molecular approaches could serve as rapid assays to predict HuNoV inactivation in high-risk food. To this end, the performance of three viability RT-qPCR assays with different intercalating markers (Viability PCR Crosslinker Kit (CL), propidium monoazide (PMAxx™), and platinum chloride (PtCl4)) in estimating survival of HuNoV exposed to thermal and high pressure (HPP) treatments was compared to replication tested in the HIE cell culture model. A nearly full-length genomic molecular assay coupled with PMAxx™ to infer HuNoV thermal inactivation was also assessed. The experimental design included HuNoV genogroup I.3 P13, GII.4 Sydney P16, GII.6 P7, along with Tulane virus (TV) serving as surrogate. Finally, viability RT-qPCR was tested in HPP-treated strawberry puree, selected as a food matrix with high viral contamination risk. PMAxx™ and CL performed evenly, while PtCl4 affected HuNoV infectivity. Taking all experimental data together, viability RT-qPCR was demonstrated to be an improved method over direct RT-qPCR to estimate viral inactivation at extreme thermal (95 °C) and HPP (450 MPa) exposures, but not under milder conditions as amplification signals were detected. Despite its complexity and limitations, the HIE demonstrated a more robust model than viability RT-qPCR to assess HuNoV infectivity.
Wales et al. (Fri,) conducted a other in Human norovirus infectivity. Viability RT-qPCR vs. Replication in human intestinal enteroids (HIE) and direct RT-qPCR was evaluated on Estimation of viral inactivation after thermal and high pressure treatments. Viability RT-qPCR improved estimation of viral inactivation at extreme thermal and high pressure exposures over direct RT-qPCR, but human intestinal enteroids provided a more robust model.