Impact of Capsid and Genomic Integrity Tests on Norovirus Extraction Recovery Rates

Abstract

Human norovirus (HuNoV) is the leading pathogen responsible for food-borne illnesses. However, both infectious and non-infectious HuNoV can be detected by RT-qPCR. This study evaluated the efficiency of different capsid integrity treatments coupled with RT-qPCR or a long-range viral RNA (long RT-qPCR) detection to reduce the recovery rates of heat inactivated noroviruses and fragmented RNA. The three capsid treatments evaluated (RNase, the intercalating agent PMAxx and PtCl4) reduced the recovery of heat inactivated HuNoV and murine norovirus (MNV) spiked on lettuce, when combined with the ISO 15216-1:2017 extraction protocols. However, PtCl4 also reduced non-heat-treated noroviruses recovery as estimated by RT-qPCR. The PMAxx and RNase treatments had a similar effect on MNV only. The most efficient approaches, the RNase and PMAxx treatments, reduced the heat-inactivated HuNoV recovery rates estimated using RT-qPCR by 2 and >3 log, respectively. The long RT-qPCR detection approach also reduced the recovery rates of heat inactivated HuNoV and MNV by 1.0 and 0.5 log, respectively. Since the long-range viral RNA amplification could be applied to verify or confirm RT-qPCR results, it also provides some advantages by reducing the risk of false positive HuNoV results.

A Multifaceted Approach for Evaluating Hepatitis E Virus Infectivity In Vitro: Cell Culture and Innovative Molecular Methods for Integrity Assessment

“…In the literature, the efficacy of RNases to degrade RNA from inactivated viruses varies, with some studies reporting successful degradation [ 44 , 58 , 60 ], while others found less favorable results [ 56 , 57 , 88 ]. These discrepancies could be due to variations in RNase concentrations, incubation times, sample types, and virus types [ 46 , 56 , 59 , 60 , 62 , 88 ].…”

“…Platinum compounds have recently been used to evaluate virion integrity of viruses such as SARS-CoV-2, norovirus, hepatitis A virus and HEV [ 41 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 ]. However, the efficiency of the applied technique varies across viruses, virus concentration, virus sample and inactivation techniques.…”

“…RNases degrade “free” RNA, thereby enhancing the likelihood of detecting viral RNA originating only from intact viral particles. This method has been tested with varying degrees of success on several viruses such as enteroviruses, rotaviruses, human norovirus, hepatitis A virus and HEV [ 44 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 ].…”

“…Successful amplification by long-range RT-qPCR suggests an intact RNA strand, indicating potential infectivity of the virus. Long-range RT-qPCR has been developed for adenovirus, murine norovirus and human norovirus [ 44 , 61 , 62 , 63 , 64 , 65 ] but has not yet been reported for HEV.…”

Spatial Distribution and Enrichment Dynamics of Foodborne Norovirus in Oyster Tissues

“…The detection of NoV in both food and environmental samples remains a significant challenge, primarily due to the complex composition of sample tissues and the low concentration of the virus present. Current human NoV cell culture systems are complicated and do not allow infectious NoV detection at levels found in contaminated foods [ 16 ]. Similarly to other viruses, detecting and monitoring NoV RNA encompasses several steps, including sample collection, concentration and enrichment, laboratory assay, and data normalization and interpretation.…”