Aims To evaluate and compare the reductions of human viruses and F‐specific coliphages in a full‐scale wastewater treatment plant based on the quantitative PCR (qPCR) and plate count assays. Methods and Results A total of 24 water samples were collected from four locations at the plant, and the relative abundance of human viruses and F‐RNA phage genogroups were determined by qPCR. Of the 10 types of viruses tested, enteric adenoviruses were the most prevalent in both influent and effluent wastewater samples. Of the different treatment steps, the activated sludge process was most effective in reducing the microbial loads. Viruses and F‐RNA phages showed variable reduction; among them, GI and GIII F‐RNA phages showed the lowest and the highest reduction, respectively. Conclusions Ten types of viruses were present in wastewater that is discharged into public water bodies after treatment. The variability in reduction for the different virus types demonstrates that selection of adequate viral indicators is important for evaluating the efficacy of wastewater treatment and ensuring the water safety. Significance and Impact of the Study Our comprehensive analyses of the occurrence and reduction of viruses and indicators can contribute to the future establishment of appropriate viral indicators to evaluate the efficacy of wastewater treatment.

Inhibitors that reduce viral nucleic acid extraction efficiency and interfere with cDNA synthesis and/or polymerase activity affect the molecular detection of viruses in aquatic environments. To overcome these significant problems, we developed a methodology for assessing nucleic acid yields and DNA amplification efficiencies for environmental water samples. This involved adding particles of adenovirus type 5 and murine norovirus and newly developed primer-sharing controls, which are amplified with the same primer pairs and result in the same amplicon sizes as the targets, to these samples. We found that nucleic acid loss during the extraction process, rather than reverse transcription-PCR (RT-PCR) inhibition, more significantly attributed to underestimation of the presence of viral genomes in the environmental water samples tested in this study. Our success rate for satisfactorily amplifying viral RNAs and DNAs by RT-PCR was higher than that for obtaining adequate nucleic acid preparations. We found that inhibitory properties were greatest when we used larger sample volumes. A magnetic silica bead-based RNA extraction method effectively removed inhibitors that interfere with viral nucleic acid extraction and RT-PCR. To our knowledge, this is the first study to assess the inhibitory properties of environmental water samples by using both control virus particles and primer-sharing controls.

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.

Presence of SARS-coronavirus-2 (SARS-CoV-2) in wastewater sample has been documented in several countries. Wastewater-based epidemiology (WBE) is potentially effective for early warning of COVID-19 outbreak. The purpose of this study was to verify the detection limit of WBE for COVID-19. In total, 27 influent wastewater samples were collected from four wastewater treatment plants in Ishikawa and Toyama prefectures in Japan. During the study period, numbers of the confirmed COVID-19 cases in these prefectures increased from almost 0 to around 20 per 100,000 peoples. SARS-CoV-2 RNA in the samples were identified by several PCR-based assays. Among the 27 samples, 7 were positive for SARS-CoV-2 by at least one out of the three quantitative RT-PCR assays. These samples were also positive by RT-nested PCR assays. The detection frequency became higher when the number of total confirmed SARS-CoV-2 cases in 100,000 peoples became above 10 in each prefecture. However, SARS-CoV-2 could also be detected with a low frequency when the number was below 1.0. Considering that the number of the confirmed cases does not necessarily reflect the actual prevalence of the infection at the time point, data on the relationship between the number of infection cases and concentration in wastewater needs to be accumulated further.