• An assessment of RT-qPCR accuracy in monitoring infectious norovirus in oyster farms

      Hunt, K.; Butler, F.; Doré, B; Keaveney, S. (University College Dublin. School of Biosystems Engineering, 2014)
      Wastewater contamination causes Norovirus (NoV) to accumulate in commercial shellfish, which is monitored using RT-qPCR. RT-qPCR does not distinguish infectious copies from non-infectious, so it is not ideal for risk assessment. Additionally, sites being sampled are assumed to be spatially homogenous, but this has not been shown. To test homogeneity, and the standard sample size of ten, a single site was intensively sampled during the 2013- 2014 winter period. Analysis of results is ongoing. Also during the winter season, in three additional sites, the ratio of infectious to noninfectious virus copies detected with RT-qPCR was modelled using an FRNA bacteriophage surrogate. Analysis of results is ongoing.
    • Characterisation of norovirus contamination in an Irish shellfishery using real-time RT-qPCR and sequencing analysis

      Rajko-Nenow, Paulina; Keaveney, Sinéad; Flannery, John; O'Flaherty, Vincent; Doré, William (Elsevier, 2012)
      Norovirus (NoV) is the single most important agent of foodborne viral gastroenteritis worldwide. Bivalve shellfish, such as oysters, grown in areas contaminated with human faecal waste may become contaminated with human pathogens including NoV. A study was undertaken to investigate NoV contamination in oysters (Crassostrea gigas) from a shellfishery over a 24 month period from October 2007 to September 2009. Oyster samples were collected monthly from a commercial shellfish harvest area classified as category B under EU regulations, but that had had been closed for commercial harvesting due to its previous association with NoV outbreaks. Real-time reverse transcription quantitative PCR (RT-qPCR) was used to determine the concentration of human NoV genogroups I and II (GI and GII) in monthly samples. Total NoV (GI and GII) concentrations in NoV positive oysters ranged from 97 to 20,080 genome copies g− 1 of digestive tissue and displayed a strong seasonal trend with greater concentrations occurring during the winter months. While NoV GII concentrations detected in oysters during both years were similar, NoV GI concentrations were significantly greater in oysters during the winter of 2008/09 than during the winter of 2007/08. To examine the NoV genotypes present in oyster samples, sequence analysis of nested RT-PCR products was undertaken. Although NoV GII.4 is responsible for the vast majority of reports of outbreaks in the community, multiple NoV genotypes were identified in oysters during this study: GI.4, GI.3, GI.2, GII.4, GII.b, GII.2, GII.12, and GII.e. NoV GI.4 was the most frequently detected genotype throughout the study period and was detected in 88.9% of positive samples, this was followed by GII.4 (43.7%) and GII.b (37.5%). This data demonstrates the diversity of NoV genotypes that can be present in sewage contaminated shellfish and that a disproportionate number of non-NoV GII.4 genotypes can be found in environmental samples compared to the number of recorded human infections associated with non-NoV GII.4 genotypes.
    • Concentration of norovirus during wastewater treatment and its impact on oyster contamination

      Flannery, John; Keaveney, Sinéad; Rajko-Nenow, Paulina; O’Flaherty, Vincent; Doré, William (American Society for Microbiology, 2012)
      Concentrations of E. coli, FRNA bacteriophage, norovirus genogroup I (NoV GI) and II (NoV GII) in wastewater were monitored weekly over a one-year period at a wastewater treatment plant (WWTP) providing secondary treatment. A total of 49 samples of influent, primary and secondary-treated wastewater were analyzed. Using a real-time RT-qPCR, mean NoV GI and NoV GII concentrations detected in effluent wastewater were 2.53 and 2.63 log10 virus genome copies 100 ml-1 respectively. Mean NoV concentrations in wastewater during the winter period (January to March inclusive) (n=12) were 0.82 (NoV GI) and 1.41 (NoV GII) log units greater than mean concentrations for the rest of the year (n=37). The mean reduction of NoV GI and GII during treatment was 0.80 and 0.92 log units respectively with no significant difference detected in the extent of NoV reductions due to season. No seasonal trend was detected in the concentrations of E. coli or FRNA bacteriophage in wastewater influent and showed mean reductions of 1.49 and 2.13 log units respectively. Mean concentrations of 3.56 and 3.72 log10 virus genome copies 100 ml-1 for NoV GI and GII respectively were detected in oysters sampled adjacent to the WWTP discharge. A strong seasonal trend was observed and concentrations of NoV GI and GII detected in oyster were correlated with concentrations detected in the wastewater effluent. No seasonal difference was detected in concentrations of E. coli or FRNA bacteriophage detected in oysters.
    • Detection of human viruses in shellfish and update on REDRISK research project, Clew Bay, Co. Mayo

      Keaveney, S.; Guilfoyle, F.; Flannery, J.; Doré, B. (Marine Institute, 2006)
      This paper describe the progress in norovirus detection methods and initial results from the REDRISK study.
    • Management of health risks associated with oysters harvested from a norovirus contaminated area, Ireland, February–March 2010

      Doré, B.; Keaveney, S.; Flannery, J.; Rajko-Nenow, P. (European Centre for Disease Prevention and Control, 2010)
      Oysters from a harvesting area responsible for outbreaks of gastroenteritis were relaid at a clean seawater site and subsequently depurated in tanks of purified seawater at elevated temperatures. This combined treatment reduced norovirus levels to those detected prior to the outbreak. On the basis of norovirus monitoring the sale of treated oysters was permitted although the harvest area remained closed for direct sale of oysters. No reports of illness have been associated with the consumption of treated oysters.
    • Norovirus genotypes present in oysters and in effluent from a wastewater treatment plant during the seasonal peak of infections in Ireland in 2010

      Rajow-Nenow, P.; Waters, A.; Keaveney, S.; Flannery, J.; Tuite, G.; Coughlan, S.; O’Flaherty, V.; Doré, W. (American Society for Microbiology, 2013)
      We determined norovirus (NoV) concentrations in effluent from a wastewater treatment plant and in oysters during the peak period of laboratory-confirmed cases of NoV infection in Ireland in 2010 (January to March). Weekly samples of influent, secondary treated effluent, and oysters were analyzed using real-time quantitative reverse transcription-PCR for NoV genogroup I (GI) and genogroup II (GII). The mean concentration of NoV GII (5.87 104 genome copies 100 ml 1) in influent wastewater was significantly higher than the mean concentration of NoV GI (1.40 104 genome copies 100 ml 1). The highest concentration of NoV GII (2.20 105 genome copies 100 ml 1) was detected in influent wastewater during week 6. Over the study period, a total of 931 laboratory-confirmed cases of NoV GII infection were recorded, with the peak (n 171) occurring in week 7. In comparison, 16 cases of NoV GI-associated illness were reported during the study period. In addition, the NoV capsid N/S domain was molecularly characterized for selected samples. Multiple genotypes of NoV GI (GI.1, GI.4, GI.5, GI.6, and GI.7) and GII (GII.3, GII.4, GII.6, GII.7, GII.12, GII.13, and GII.17), as well as 4 putative recombinant strains, were detected in the environmental samples. The NoV GII.4 variant 2010 was detected in wastewater and oyster samples and was the dominant strain detected in NoV outbreaks at that time. This study demonstrates the diversity of NoV genotypes present in wastewater during a period of high rates of NoV infection in the community and highlights the potential for the environmental spread of multiple NoV genotypes.
    • Preliminary results from a survey of oyster production areas in Ireland for norovirus

      Keaveney, S.; Flannery, J.; Guilfoyle, F.; Doré, J. (2007)
      A survey of 18 oyster production areas in Ireland for norovirus (NoV) contamination was initiated in August 2006. The findings presented are the preliminary results from the first seven months of the survey. Prior to the survey commencing, a simple desk bask sanitary survey of each area was undertaken. This provided an assessment enabling each site to be ranked into 3 categories (low, medium and high) on the basis of the risk of NoV contamination. Samples were collected on a monthly basis and tested for the presence of NoV using semi-quantitative real-time PCR allowing relative quantitation of NoV levels. A correlation was observed between occurrence and levels of NoV detected and the risk categories ascribed to each production area. To date NoV was detected in 60.7, 30.0 and 2.5 percent of samples from the high, medium and low risk categorised areas, respectively. A strong seasonal bias towards increased winter contamination was observed with NoV detected in 15.5 and 50 % of samples in August and February, respectively. The preliminary results from this survey indicate that it may be possible to predict the relative risk of NoV contamination in a shellfish harvesting area. This in conjunction with targeted NoV monitoring using real-time PCR could aid the further development of risk management procedures in shellfisheries.
    • REDRISK: reduction of the virus risk in shellfish harvesting areas

      Guilfoyle, F.; Keaveney, S.; Flannery, J.; Doré, B. (Marine Institute, 2006)
      Filter feeding bivalve shellfish can accumulate human pathogenic bacteria and viruses if grown in sewage-contaminated waters. Current consumer protection legislation relies on classification of harvesting areas based on their sanitary quality, using E coli as an indicator of sewage contamination. Advances in viral monitoring have shown that E coli can underestimate the extent of the contamination. The most common cause of gastroenteritis associated with shellfish is norovirus, commonly known as winter vomiting virus. The REDRISK project was undertaken to investigate the main environmental factors that cause viral contamination in shellfish. The REDRISK project is part of a EU research pillar with parallel research being undertaken in the UK, France and Spain. A recently developed technique to quantify norovirus in shellfish, real-time PCR, has been used in the REDRISK project. Clew Bay, in Co. Mayo was chosen as the study area in Ireland. The bay is generally considered to have good water quality but with certain areas subject to intermittent sewage contamination. The cooperation of local producers and organisations such as the Clew Bay Marine Forum and the Native Oyster Co-op greatly helped the project. The project was divided into a two-phased approach. Phase one involved the identification of contamination sources impacting the bay through a sanitary survey and selection of appropriate sites for further study. Results of the first phase of this study were presented previously at this forum (Keaveney, et al 2006) and the characteristics of the sites selected for study and locations within the bay are shown in table 1 and figure 1 respectively. The second phase of the project focused on monitoring environmental conditions and microbiological levels in shellfish to identify environmental conditions leading to viral contamination. This paper reports the finding of this monitoring.
    • Simulated sunlight inactivation of norovirus and FRNA bacteriophage in seawater

      Flannery, J.; Rajko-Nenow, P.; Keaveney, S.; O'Flaherty, V.; Doré, W. (Wiley, 2013)
      Aims: To investigate norovirus (NoV) and F-specific RNA (FRNA) bacteriophage inactivation in seawater under simulated sunlight and temperature conditions representative of summer (235 W m−2; 17°C) and winter (56 W m−2; 10°C) conditions in Ireland. Methods and Results: Inactivation experiments were carried out using a collimated beam of simulated sunlight and 100 ml of filtered seawater seeded with virus under controlled temperature conditions. NoV concentrations were determined using RT-qPCR, and FRNA bacteriophage concentrations were determined using RT-qPCR and by plaque assay. For all virus types, the fluence required to achieve a 90% reduction in detectable viruses (S90 value) using RT-qPCR was not significantly different between summer and winter conditions. S90 values for FRNA bacteriophage determined by plaque assay were significantly less than those determined by RT-qPCR. Unlike S90 values determined by RT-qPCR, a significant difference existed between summer and winter S90 values for infectious FRNA bacteriophage. Conclusions: This study demonstrated that RT-qPCR significantly overestimates the survival of infectious virus and is therefore unsuitable for determining the inactivation rates of viruses in seawater. Significance and Impact of the Study: Results from this study provide initial data on the inactivation of NoV and FRNA bacteriophage in seawater under representative summer and winter conditions and will be of interest to shellfish and water management agencies alike.