Spatial and temporal distributions of enteric viruses and indicators in a lake receiving municipal wastewater treatment plant discharge.

Although lake water can be used as a source of drinking water and recreational activities, there is a dearth of research on the occurrence and fate of enteric viruses. Over a period of 14 months at six points in 2014-2015, we conducted monthly monitoring of the virological water quality of a Japanese lake. The lake receives effluent from three surrounding wastewater treatment plants and retains water for about two weeks. These features allowed us to investigate the occurrence and fate of viruses in the lake environment. Human enteric viruses such as noroviruses and their indicators (pepper mild mottle virus and F-specific RNA bacteriophage [FRNAPH] genogroups) were quantified by PCR-based assays. Additionally, FRNAPH genogroups were quantified by infectivity-based assays to estimate the degree of virus inactivation. Pepper mild mottle virus, genogroup II (GII) norovirus, and GI-FRNAPH were identified in relatively high frequencies (positive in >40% out of 64 samples), with concentrations ranging from 1.3 × 101 to 2.9 × 104 copies/L. Human enteric viruses and some indicators were not detected and less prevalent, respectively, after April 2015. Principal component analysis revealed that the virological water quality changed gradually over time, but its differences between the sampling points were not apparent. FRNAPH genogroups were inactivated during the warm season (averaged water temperature of >20 °C) compared to the cool season (averaged water temperature of <20 °C), which may have been due to the more severe environmental stresses such as sunlight and water temperature. This suggests that the infection risk associated with the use of the lake water may have been overestimated by the gene quantification assay during the warm season.

Comprehensive Study on Enteric Viruses and Indicators in Surface Water in Kyoto, Japan, During 2014-2015 Season.

Certain enteric viruses that are present in the water environment are potential risk factors of waterborne infections. To better understand the impact of viruses in water, both enteric viruses and their potential indicators should be comparatively investigated. In this study, occurrences of GI- and GII-noroviruses (NoVs), sapovirus (SaV), rotavirus (RoV), Aichi virus 1 (AiV-1), enterovirus (EV), and pepper mild mottle virus (PMMoV) were quantitatively determined in surface water samples in Japan. Additionally, the genotype distribution of GI- and GII-NoVs was determined using a next-generation amplicon sequencing. PMMoV was the most abundant virus regardless of season and location, indicating its usefulness as an indicator for the viral contamination of water. Other potential indicators, AiV and EV, were less abundant than GII-NoV. Viruses other than PMMoV showed seasonality, i.e., EV and other viruses (NoVs, SaV, RoV, and AiV-1) became prevalent during summer and winter, respectively. SaV showed a relatively high abundance at a location that was affected by untreated wastewater. Regarding NoV genotypes, GI.1, GI.2, GI.4, GI.5, GI.6, GII.3, GII.4, GII.6, and GII.17 were found from the surface water samples. GII.4 and GII.17 seemed to have contributed to the high abundance of GII-NoV in the samples. Interestingly, GII.17 strains became prevalent in the water samples before becoming prevalent among gastroenteritis patients in Japan. These findings provide further insights into the properties of viruses as contaminants in the water environment.

Quantitative Distribution of Infectious F-Specific RNA Phage Genotypes in Surface Waters.

UNLABELLED: F-specific RNA phages (FRNAPHs) are considered potential viral indicators of water pollution due to their occurrence and stability in water environments. However, their suitability as viral indicators is not fully elucidated because the characteristics of FRNAPHs are variable depending on the genotype. In this study, for the characterization of infectious FRNAPH genotypes, integrated culture reverse transcription-PCR coupled with the most probable number approach was applied to surface water samples. Further, to recover low concentrations of FRNAPH genotypes, an FRNAPH recovery method was developed. The novel FRNAPH recovery method using a noncharged microfiltration membrane could effectively recover FRNAPH strains without inactivation, while a method using an electronegative microfiltration membrane resulted in the inactivation of some strains. Infectious FRNAPH genotypes in surface water samples were successfully quantified with an efficiency comparable to that of the conventional plaque assay. Genotype I (GI) and GII FRNAPHs tended to be predominant at locations impacted by treated and untreated municipal wastewater, respectively. The numbers and proportions of infectious FRNAPHs tended to be higher during the winter season when water temperature decreased. IMPORTANCE: Properties of FRNAPHs are highly variable depending on their genotypes. Previous typing methods for FRNAPHs are not quantitative and/or are based on molecular assays, which cannot differentiate infective strains from inactive strains. Due to the reasons mentioned above, the utility of FRNAPHs as viral indicators of water pollution has not been fully validated. In this study, a quantitative genotyping method for infectious FRNAPHs was developed and applied to surface water samples. The method enabled characterization of infectious FRNAPH genotypes in terms of their occurrence and seasonality. Moreover, comparison of the method to a conventional molecular assay (reverse transcription-quantitative PCR) enabled characterization of their stability. Our approach can provide novel findings for further validation of FRNAPHs as viral indicators of water pollution.