Pepper mild mottle virus intended for use as a process indicator for drinking water treatment: Present forms and quantitative relations to norovirus and rotavirus in surface water.

Pepper mild mottle virus (PMMoV) has been proposed as a potential indicator of human enteric viruses in environmental water and for viral removal during drinking water treatment. To investigate the occurrence and present forms of PMMoV and quantitative relations to norovirus GII and rotavirus A (RVA) in surface waters, 147 source water samples were collected from 21 drinking water treatment plants (DWTPs) in Japan between January 2018 and January 2021, and the concentrations of viruses in suspended and dissolved fractions were measured using real-time RT-PCR. PMMoV was detected in 81-100 % of samples in each sample month and observed concentrations ranged from 3.0 to 7.0 log10 copies/L. The concentrations of PMMoV were higher in dissolved fraction compared to suspended fractions, while different partitioning was observed for NoV GII depending on seasons. The concentrations of PMMoV were basically higher than those of norovirus GII (1.9-5.3 log10 copies/L) and RVA (1.9-6.6 log10 copies/L), while in 18 samples, RVA presented higher concentrations than PMMoV. Partial regions of VP7, VP4, and VP6 of the RVA in the 18 samples were amplified using nested PCR, and the genotypes were determined using an amplicon-based next-generation sequencing approach. We found that these source water samples included not only human RVA but also various animal RVA and high genetic diversity due to the existence of animal RVA was associated with a higher RVA concentration than PMMoV. Our findings suggest that PMMoV can be used as an indicator of norovirus GII and human RVA in drinking water sources and that the indicator performance should be evaluated by comparing to zoonotic viruses as well as human viruses.

Temporal variations of human and animal Rotavirus A genotypes in surface water used for drinking water production.

Rotavirus is a major cause of gastroenteritis among infants and children. In this study, nested PCR assays were developed to amplify partial regions of the VP7, VP4, and VP6 genes of Rotavirus A (RVA) for amplicon-based Illumina MiSeq sequencing to investigate RVA genotypes in environmental water samples. Eight sets of inner primers were first designed and screened for use in the nested PCR assays, and four sets of them could produce amplicons. Six sets of outer primers were then designed and combined with the four sets of inner primers that worked. The assays were evaluated for sensitivity using raw water samples collected from one drinking water treatment plant between April 2019 and March 2020 (Sample Set 1; N = 12) and seven DWTPs between 2018 and 2020 (Sample Set 2; N = 18). In total, 43 amplicons from Set 1 were sequenced and diverse sequences from human, porcine, bovine, equine, and feline RVA were observed. Human G8, G3, and G2 genotypes were obtained, with G8 predominant (relative abundance, 36-87%) in samples taken during the rotavirus epidemic season between April and June. Porcine G5, G11, and G4, and bovine G10 and G6 genotypes were also detected. VP4 sequence analysis revealed that the human P[8] genotype was present throughout the year, whereas P[4] and P[9] were present only in the epidemic season. The vaccine strains P[5] and P[8] (RotaTeq®) were also detected. Our approach enables the identification of prevalent human and animal RVA genotypes and their host species that potentially caused fecal contamination in water sources.

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.