1 National Food Institute, Technical University of Denmark2 Swedish Institute for Infectious Disease Control3 Norwegian School of Veterinary Science
Noroviruses (NoVs) are the principal cause of viral gastroenteritis worldwide. NoVs are extremely infectious with an estimated infectious dose 50% (ID50) of 18 viral particles. Transmission of NoVs occurs via the faecal-oral route directly through person-to-person contact or indirectly via contaminated fomites, water and food. In recent years, numerous outbreak of NoV-associated gastroenteritis have been linked to the consumption of contaminated drinking water, shellfish and fresh produce. In addition, indications of transmission occurring though aerosol formation, especially following projectile vomiting, have been seen, but little is at present known about the possibility and extent of airborne transmission of NoVs. To reduce transmission of NoV it is imperative to have efficient virus recovery methods available that can be used for routine analysis of the implicated matrices. However, currently no standardised procedures exist for this purpose. The aim of this PhD thesis was to develop and evaluate methods for recovery of NoV from shellfish and drinking water that could contribute to the work done towards finding suitable standardised methods. This was done through three studies, presented in manuscript I-III. Additionally, exposure to airborne NoV transmission and NoV decontamination of food surfaces were addressed in manuscript IV and V, respectively. In manuscript I five methods were compared for the ability to qualitatively recover NoV GII and feline calicivirus (FCV) spiked in the digestive tissue of oysters and blue mussels. A method based proteinase K digestion followed by NucliSens miniMAG nucleic acid extraction was found to give the best performance. In a subsequent collaborative trial the method was found to robustly recover the NoV GI, NoV GII and HAV bioaccumulated in the both oysters and mussels. Consequently, the method was found to be a good candidate as a future qualitative standard for routine viral analysis of oysters and mussels. The ability of a rapid method to recover NoV GI, NoV GII and adenovirus (AdV) from Nordic drinking water (tap water) of various types was evaluated in manuscript II. The method was based on filtering using a positively charged membrane followed by direct lysis of the virus adsorbed to the membrane. The average efficiency of the method to recover NoV GI (43±29%) and GII (45±24%) from drinking water was generally found to be better, or at least comparable, to previously published methods, suggesting that the method could be suitable for routine analysis. However, virus and water type were found to significantly affect the recovery. As an alternative strategy for the recovery of NoVs from drinking water the feasibility of using virus imprinted polymers (VIPs) for capture and selective recognition of NoVs was investigated in manuscript III. Three VIPs targeted for murine norovirus (MNV), NoV GI and NoV GII, respectively, were synthesised and experiments to determine binding of the target viruses and a non-target AdV to the VIPs and to non-imprinted polymers (NIPs) were conducted. Unfortunately, a relative poor selective recognition and high degree of unspecific binding to the VIPs were observed. Thus, VIPs in their current form do not appear to be an optimal approach for recovery of NoVs from drinking water. Aerosolisation of microorganisms may occur during treatment of wastewater. Therefore, the exposure to aerosolised NoVs and other bioaerosol components at a wastewater treatment plant (WWTP) was examined using personal samples in manuscript IV. NoVs were, for the first time, detected on a dust filter carried by a WWTP worker in concentrations that could pose an occupation risk. Because the workers at the WWTP had previously been found to have an increase frequency of acute gastrointestinal illness, consistent with NoV infection, our finding suggest that that airborne transmission of NoVs do indeed occur at the WWTP. Finally, manuscript V evaluated the efficiency of a surface decontamination strategy based on combined treatment with steam and ultrasound to inactivate the NoV surrogates FCV, MNV and bacteriophage MS2. The steam-ultrasound treatment was found to be effective for decontamination of plastic surfaces with near complete inactivation observed after treatment for 1 or 3 s, depending on the NoV surrogate. However, steam-ultrasound treatment in it current form was found to be inappropriate for NoV decontamination of raspberries as only 1-log reduction of MS2 was achieved after treatment 1 s at which point unwanted loss in the texture of the raspberries were observed.