Recovery of Nucleic Acids of Enteric Viruses and Host-Specific Bacteroidales from Groundwater by Using an Adsorption-Direct Extraction Method.

In this study, the adsorption-elution method was modified to concentrate viral particles in water samples and investigate the contamination of groundwater with norovirus genogroup II (NoV GII), rotavirus A (RVA), and Pepper mild mottle virus (PMMoV). The mean recovery rate of a murine norovirus strain, which was inoculated into groundwater samples collected from a deep well, was the highest (39%) when the viral RNA was directly extracted from the membrane instead of eluting the adsorbed viral particles. This adsorption-direct extraction method was applied to groundwater samples (20 liters) collected from deep wells used for the public drinking water supply (n = 22) and private wells (n = 9). RVA (85 copies/liter) and NoV GII (35 copies/liter) were detected in water samples from a deep well and a private well, respectively. PMMoV was detected in 95% and 89% of water samples from deep wells and private wells, respectively, at concentrations of up to 990 copies/liter. The modified method was also used to extract bacterial DNA from the membrane (recovery rate of inoculated Escherichia coli K-12 was 22%). The Bacteroidales genetic markers specific to ruminants (BacR) and pigs (Pig2Bac) were detected in samples from a deep well and a private well, respectively. The modified virus concentration method has important implications for the management of microbiological safety in the groundwater supply. IMPORTANCE We investigated the presence of enteric viruses and bacterial genetic markers to determine fecal contamination in groundwater samples from deep wells used for the public drinking water supply and private wells in Japan. Groundwater is often subjected to chlorination; malfunctions in chlorine treatment result in waterborne disease outbreaks. The modified method successfully concentrated both viruses and bacteria in 20-liter groundwater samples. Norovirus genogroup II (GII), rotavirus A, Pepper mild mottle virus, and Bacteroidales genetic markers specific to ruminants and pigs were detected. Frequent flooding caused by increased incidences of extreme rainfall events promotes the infiltration of surface runoff containing livestock wastes and untreated wastewater into wells, possibly increasing groundwater contamination risk. The practical and efficient method developed in this study will enable waterworks and the environmental health departments of municipal/prefectural governments to monitor water quality. Additionally, the modified method will contribute to improving the microbiological safety of groundwater.

Detection of Norovirus and Rotavirus Present in Suspended and Dissolved Forms in Drinking Water Sources.

We investigated the present forms of genogroup II norovirus and group A rotavirus in surface water used for drinking water production. River water samples (N = 15) collected at a drinking water treatment plant (DWTP) monthly from June 2017 to August 2018 were fractioned by filtration through 10- and 0.45-µm-pore-size membranes, and viruses present in suspended and dissolved forms were quantitatively detected. Norovirus GII was present in > 10-µm- and 0.45-10-µm-suspended and dissolved forms with detection rates of 33%, 60%, and 87%, respectively. Rotavirus A was detected more frequently than norovirus GII in each form (> 10 µm suspended, 73%; 0.45-10 µm suspended, 93%; dissolved, 100%). We also analyzed surface water samples from 21 DWTPs all over Japan in non-epidemic and epidemic seasons of gastroenteritis. Norovirus GII was detected in 48% and 81% of samples with the concentrations of up to 4.1 and 5.3 log10 copies/L in dissolved form in non-epidemic and epidemic seasons, respectively, and GII.4 Sydney 2012 was predominant genotype followed by GII.2. Rotavirus A was detected in 95% and 86% of samples with the maximum concentrations of 5.5 and 6.3 log10 copies/L in dissolved form in respective seasons. Concentration of norovirus GII was similar in 0.45-10-µm suspended and dissolved forms, while there was a significant difference for rotavirus A (P < 0.01, pared t test), indicating that rotavirus A was less associated with suspended solids in the surface water samples compared to norovirus GII. Our observations provide important implications for understanding of viral behavior in environmental waters.