The Public Health Implications of Real Time PCR Detection of Norovirus in Bivalve Molluscan Shellfish.

• Main Author: Dancer, Daniel.
• Format: Thesis
• Language: English
• Published: University of Surrey 2011
• Subjects: Pacific; Crassostrea gigas
• Online Access: http://epubs.surrey.ac.uk/854959/1/27558131.pdf

Norovirus (NoV) cannot be routinely cultured in the laboratory and therefore existing methods for detection are based on molecular assays. Application of these assays has shown that a high percentage of bivalve molluscan shellfish (BMS) production areas in the UK are contaminated with NoV. It is unclear whether detection of viral genome always correlates with presence of whole infectious virus and thus public health risk. This project aimed to investigate the significance of detecting norovirus in the environment and in BMS by real-time PCR. Murine norovirus (MNV), a closely related NoV, was used as a surrogate in experimental work to determine the behaviour of NoV in the environment and in the BMS matrix in terms of viability and infectivity. A real-time PCR assay for MNV was developed and characterised, and was determined to be equivalent to the in-house real-time PCR assay for human NoV at this laboratory. This assay was then used alongside an existing cell culture method for MNV that was adopted and optimised in this project. The development of a plaque assay with both sufficient robustness and sensitivity for application to bioaccumulated oysters was unsuccessful. These methods, and existing methods for human NoV were used to evaluate environmental persistence of the human NoV genome, human NoV and its surrogate in seawater under artificial environmental conditions simulating winter in the United Kingdom (U. K). The ability of Pacific oysters (Crassostrea gigas) to bioaccumulate both untreated and heat treated NoV particles (from human faeces) and RNA fragments was compared using real-time PCR. Finally, the potential application and incorporation of a pre-extraction treatment into the BMS testing method was assessed. The results presented in this project indicated that NoV RNA may reside in seawater for up to 2 weeks, and that there was no significant difference between the rates of degradation in seawater of naked RNA transcripts and NoV particles from faecal material. However MNV-1 persisted longer in seawater than human NoV under the same conditions. The results presented also indicated that BMS bioaccumulate NoV RNA fragments signifcantly less efficiently than NoV particles from faecal material. Furthermore, the uptake of untreated human NoV and MNV occurred at similar rates to the uptake of heat-treated viruses. It was also determined that using RNaseONE as a pre-extraction treatment to remove PCR signal from ‘free’ RNA and damaged viruses provided scope for the inclusion into the BMS testing method to provide a better prediction of human health risk, however further work would be required to demonstrate its efficacy in a standardised method.