Human astroviruses are associated with gastroenteritis and known to contaminate water environments. Three different genetic clades of astroviruses are known to infect humans and each clade consists of diverse strains. This study aimed to determine the occurrence and genetic diversity of astrovirus strains in water samples in different geographical locations, i.e., influent and effluent wastewater samples (n = 24 each) in Arizona, U.S., and groundwater (n = 37) and river water (n = 14) samples collected in the Kathmandu Valley, Nepal, using next-generation amplicon sequencing. Astrovirus strains including rare types (types 6 and 7 classical human astroviruses), emerging type (type 5 VA-astroviruses), and putative recombinants were identified. Feline astrovirus strains were collaterally identified and recombination between human and feline astroviruses was suggested. Classical- and VA-astroviruses seemed to be prevalent during cooler months, while MLB-astroviruses were identified only during warmer months. This study demonstrated the effectiveness of next-generation amplicon sequencing for identification and characterization of genetically diverse astrovirus strains in environmental water.

Evaluating the reduction of virus load in water reclamation plants is important to ensuring the hygienic safety of the reclaimed water. A virus-spiking test is usually used to estimate virus reduction but is not practicable at large-scale plants. Thus, we evaluated virus reduction by ultrafiltration (UF) plus ultraviolet (UV) irradiation at a large-scale reclamation plant (1000 m³/d) by quantifying indigenous F-specific RNA bacteriophages (FRNAPHs). To detect the infectious FRNAPH, we used both plaque assay and integrated culture–reverse-transcription polymerase chain reaction combined with the most probable number assay, which can detect infectious FRNAPH genotypes. For comparison, we determined reductions of indigenous FRNAPHs and spiked MS2 at a small-scale pilot plant (10 m³/d) at the same time. Reductions by UF were not significantly different among the bacteriophages at pilot plants. This result suggests that indigenous bacteriophages could be used for evaluating virus reduction by UF at large-scale plants. Indigenous Genotype I (GI) FRNAPH showed the highest UV resistance, followed by GII, GIII, and GIV. The resistance of GI-FRNAPH was equivalent to that of spiked MS2. The reduction of the total infectious FRNAPHs determined by plaque assay was affected by the predominant FRNAPH genotype, presumably because of their different UV resistances. Our results reveal that indigenous GI-FRNAPH can be a good alternative indicator to spiked MS2 in view of virus reduction during water reclamation. The reclaimed water from our large-scale reclamation plant could be used for irrigation because the expected reduction (6.3 log₁₀) of indigenous GI-FRNAPH achieved the Title 22 (>5 log₁₀).

F-specific RNA bacteriophages (FRNAPHs) have been suggested as good indicators of the presence of human enteric viruses in water treatment facilities. The occurrence and reduction of norovirus (NoV) and FRNAPH genotypes in wastewater treatment plants (WWTPs) have been well studied; however, the relationship between these genotypes in WWTPs has not been fully elucidated. Thus, we aimed to investigate the occurrence and reduction of FRNAPH genotypes in an attempt to identify NoV indicators in a WWTP via a 1-year survey. All FRNAPH and NoV genotypes were detected in WWTP influents at high rates (71–100%), including the infectious FRNAPH genotype IV (GIV), which has been rarely detected in previous studies. The reductions of FRNAPH GII and NoV GII during wastewater treatment indicated a relationship between the two (r = 0.69, P < 0.01), and the mean values were not significantly different. These results suggested that FRNAPH GII could be used as an appropriate indicator of NoV GII during wastewater treatment. FRNAPH GI was also found to be an appropriate indicator of viral reduction because of its high resistance to wastewater treatment compared with the other FRNAPH and NoV genotypes; therefore, it can be considered as a worst-case scenario organism.