The development of LENTICULES™ as reference materials for noroviruses.

AIMS: To investigate the potential for LENTICULES™ to act as reference materials (RMs) for noroviruses (NoV) [genogroups I (GI) and II (GII)] by determining their homogeneity and stability characteristics. METHODS AND RESULTS: NoV used in this study originated from human faecal material, screened for the absence of other faecally transmitted pathogens. The norovirus strains present in the faecal material were characterized by sequencing, and samples containing GI and GII strains representative of genotypes commonly circulating in the community were selected. RMs were produced utilizing modified lenticulating technology. A batch comprising 500 LENTICULES™ containing both norovirus genogroups was produced according to ISO Guide 34. The batch was tested and quantified using an ISO 17025 accredited quantitative real-time RT-PCR assay. Sufficient homogeneity was established using procedures described by Fearn and Thompson (2010), while stability at less than -15°C and ambient temperature (17-22°C) was assessed over 52 weeks and 7 days, respectively. CONCLUSIONS: Lenticulation was shown to be an effective means of preservation of detectable NoV. LENTICULES™ were sufficiently homogeneous and stable throughout medium-term frozen and short-term storage at room temperature to serve as RMs. Virus LENTICULES™ have the advantages of being easy to manipulate, provide assigned values and do not require the manipulation of high titre clinical material. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study show that norovirus LENTICULES™ can be used as stable RMs for quantitative real-time RT-PCR assays. They can be utilized as in-run positive extraction controls and potentially for method calibration and to enable more easy comparison of data generated by the variety of differing norovirus determination methods that have emerged in recent years. LENTICULES™ have the potential to provide essential elements of laboratory quality assurance systems for laboratories implementing these new methods for virus testing in foodstuffs and for those running routine analyses.

Human norovirus RNA persists in seawater under simulated winter conditions but does not bioaccumulate efficiently in Pacific Oysters (Crassostrea gigas).

Norovirus (NoV) is the principal agent of bivalve molluscan shellfish-associated gastroenteric illness worldwide. Currently, noncultivable human NoVs can be detected in bivalve molluscan shellfish by using molecular methods such as real-time reverse transcription PCR assays (qRT-PCR). In addition to infectious viruses, this methodology may also detect noninfectious NoV, including fragments of the NoV genome. This study addresses, in part, the implications of qRT-PCR results for the detection of NoV in shellfish in the absence of an infectivity assay. To evaluate environmental persistence, the stability of a short fragment of the NoV genome, spanning the qRT-PCR target in the open reading frame 1/2 junction, was assessed in seawater under artificial environmental conditions simulating winter in the United Kingdom (1 mW/cm² UV irradiation, 8°C) during a 4-week period. Detectable RNA levels decreased exponentially (T90 of approximately 141 h); however, sequences were still detectable for up to 2 weeks. The ability of Pacific oysters (Crassostrea gigas) to bioaccumulate NoV particles (from human feces) and RNA fragments was also compared using qRT-PCR. Oysters exposed to NoV particles subsequently were positive for NoV by qRT-PCR at levels several orders of magnitude in excess of the theoretical limit of detection, whereas oysters exposed to similar quantities of NoV RNA were either negative or positive at significantly lower levels. Therefore, although noninfectious fragments of NoV RNA may persist in the environment under winter conditions, this type of material will not be efficiently bioaccumulated by Pacific oysters and should not significantly contribute to positive qRT-PCR results.