ABSTRACTThe concentrations ofEscherichia coli, F-specific RNA bacteriophage (FRNA bacteriophage), and norovirus genogroup I (NoV GI) and norovirus genogroup II (NoV GII) in wastewater were monitored weekly over a 1-year period at a wastewater treatment plant (WWTP) providing secondary wastewater treatment. A total of 49 samples of influent wastewater and wastewater that had been treated by primary and secondary wastewater treatment processes (primary and secondary treated wastewater) were analyzed. Using a real-time reverse transcription-quantitative PCR (RT-qPCR), the mean NoV GI and NoV GII concentrations detected in effluent wastewater were 2.53 and 2.63 log10virus genome copies 100 ml−1, respectively. The mean NoV concentrations in wastewater during the winter period (January to March) (n= 12) were 0.82 (NoV GI) and 1.41 (NoV GII) log units greater than the mean concentrations for the rest of the year (n= 37). The mean reductions of NoV GI and GII during treatment were 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 ofE. colior 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 log10virus genome copies 100 ml−1for NoV GI and GII, respectively, were detected in oysters sampled adjacent to the WWTP discharge. A strong seasonal trend was observed, and the concentrations of NoV GI and GII detected in oysters were correlated with concentrations detected in the wastewater effluent. No seasonal difference was detected in concentrations ofE. colior FRNA bacteriophage detected in oysters.
This review aims to assess and recommend approaches for targeted and agnostic High Throughput Sequencing of RNA viruses in a variety of sample matrices. HTS also referred to as deep sequencing, next generation sequencing and third generation sequencing; has much to offer to the field of environmental virology as its increased sequencing depth circumvents issues with cloning environmental isolates for Sanger sequencing. That said however, it is important to consider the challenges and biases that method choice can impart to sequencing results. Here, methodology choices from RNA extraction, reverse transcription to library preparation are compared based on their impact on the detection or characterization of RNA viruses.
Male-specific (F) RNA bacteriophages have been proposed as indicators for human enteric viruses in shellfish. This study compared the use of Escherichia coli and FRNA bacteriophages to indicate the presence and level of noroviruses in Crassostrea gigas. A total of 167 samples from category A and B shellfish harvesting areas were analyzed for E. coli and FRNA bacteriophages by standard methods and for noroviruses (NoV) by using a previously described real-time PCR assay. FRNA bacteriophage and NoV levels in shellfish showed a seasonal trend and were elevated during the winter period (October through March). Conversely, E. coli levels did not reflect this seasonal trend. Categorizing samples on the basis of E. coli levels according to European Union regulatory limits failed to indicate the occurrence or level of NoV in shellfish. However, by grouping shellfish samples on the basis of FRNA bacteriophage levels a clear correlation was observed with NoV levels. The use of FRNA bacteriophages to predict the occurrence of NoV in shellfish could provide improved public health protection and should be considered when developing risk management procedures for shellfisheries.
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 × 104genome copies 100 ml−1) in influent wastewater was significantly higher than the mean concentration of NoV GI (1.40 × 104genome copies 100 ml−1). The highest concentration of NoV GII (2.20 × 105genome 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.
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