Activity- and gene-based quantification of enteric viruses, F- specific RNA phage genogroups, pepper mild mottle virus, and Escherichia coli in surface water.

Polymerase chain reaction (PCR) is widely applied for the monitoring of pathogenic viruses in water environments. To date, several pretreatments to selectively detect genes from infectious viruses via PCR have been developed. This study was aimed to characterize and validate methods for quantifying active viruses and indicators and to evaluate the proportion of their active fractions in surface water (n = 42). Active E. coli and F-specific RNA phage (FRNAPH) genogroups were quantified using culture assays. In addition to these microbes, norovirus genogroups I (GI) and II, Aichi virus 1, and pepper mild mottle virus (PMMoV) were quantified by (reverse transcription)-quantitative PCR (RT-qPCR) with and without cis-dichlorodiammineplatinum (CDDP) treatment to exclude genes in inactive viruses. CDDP-RT-qPCR showed concentrations and detection frequencies comparable to or higher than culture assays. Consequently, although CDDP-RT-qPCR can suggest the presence of an inactive virus, it can also overestimate the activity of the virus in the environment. Differences between culture and CDDP-RT-qPCR and between CDDP-RT-qPCR and RT-qPCR varied among the viruses. CDDP-RT-qPCR showed a concentration comparable to the culture assay (within 1 log10 difference) in 93 % of positive samples for GI-FRNAPH but in <63 % of positive samples for GII- and GIII-FRNAPHs. GII-NoV was detected from 5 and 30 out of 42 samples via CDDP-RT-qPCR and RT-qPCR, respectively, and was suggested as inactivated by 2.0 log10 or higher in most of the samples. By contrast, concentrations of PMMoV determined by these two assays were not notably different. It is suggested that the operational conditions of wastewater treatment plants around the sites, rather than environmental stresses, affected the microbial inactivation. To better understand the infectivity of viruses in the environment, it is important to investigate them using sensitive detection methods at various sites, including the source of contamination.

Surfactant Treatment for Efficient Gene Detection of Enteric Viruses and Indicators in Surface Water Concentrated by Ultrafiltration.

The hollow fiber ultrafiltration (HFUF)-based microbial concentration method is widely applied for monitoring pathogenic viruses and microbial indicators in environmental water samples. However, the HFUF-based method can co-concentrate substances that interfere with downstream molecular processes-nucleic acid extraction, reverse transcription (RT), and PCR. These inhibitory substances are assumed to be hydrophobic and, therefore, expected to be excluded by a simple surfactant treatment before the silica membrane-based RNA extraction process. In this study, the efficacy and limitations of the sodium deoxycholate (SD) treatment were assessed by quantifying a process control and indigenous viruses using 42 surface water samples concentrated with HFUF. With some exceptions, which tended to be seen in samples with high turbidity (> 4.0 NTU), virus recovery by the ultrafiltration method was sufficiently high (> 10%). RNA extraction-RT-quantitative PCR (RT-qPCR) efficiency of the process control was insufficient (10%) for 30 of the 42 HFUF concentrates without any pretreatments, but it was markedly improved for 21 of the 30 inhibitory concentrates by the SD treatment. Detection rates of indigenous viruses were also improved and no substantial loss of viral RNA was observed. The SD treatment was particularly effective in mitigating RT-qPCR inhibition, although it was not effective in improving RNA extraction efficiency. The methodology is simple and easily applied. These findings indicate that SD treatment can be a good alternative to sample dilution, which is widely applied to mitigate the effect of RT-qPCR inhibition, and can be compatible with other countermeasures.

Applicability of F-specific bacteriophage subgroups, PMMoV and crAssphage as indicators of source specific fecal contamination and viral inactivation in rivers in Japan.

To date, several microbes have been proposed as potential source-specific indicators of fecal pollution. 16S ribosomal RNA gene markers of the Bacteroidales species are the most widely applied due to their predominance in the water environment and source specificity. F-specific bacteriophage (FPH) subgroups, especially FRNA phage genogroups, are also known as potential source-specific viral indicators. Since they can be quantified by both culture-based and molecular assays, they may also be useful as indicators for estimating viral inactivation in the environment. Pepper mild mottle virus (PMMoV) and crAssphage, which are frequently present in human feces, are also potentially useful as human-specific indicators of viral pollution. This study aimed to evaluate the applicability of FPH subgroups, PMMoV, and crAssphage as indicators of source-specific fecal contamination and viral inactivation using 108 surface water samples collected at five sites affected by municipal and pig farm wastewater. The host specificity of the FPH subgroups, PMMoV, and crAssphage was evaluated by principal component analysis (PCA) along with other microbial indicators, such as 16S ribosomal RNA gene markers of the Bacteroidales species. The viabilities (infectivity indices) of FRNA phage genogroups were estimated by comparing their numbers determined by infectivity-based and molecular assays. The PCA explained 58.2% of the total information and classified microbes into three groups: those considered to be associated with pig and human fecal contamination and others. Infective and gene of genogroup IV (GIV)-FRNA phage were assumed to be specific to pig fecal contamination, while the genes of GII-FRNA phage and crAssphage were identified to be specific to human fecal contamination. However, PMMoV, infective GI-FRNA phage, and FDNA phage were suggested to not be specific to human or pig fecal contamination. FRNA phage genogroups, especially the GIV-FRNA phage, were highly inactivated in the warm months in Japan (i.e., July to November). Comparing the infectivity index of several FRNA phage genogroups or other viruses may provide further insight into viral inactivation in the natural environment and by water treatments.

Detection of SARS-CoV-2 in wastewater in Japan during a COVID-19 outbreak.

The presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in wastewater samples has been documented in several countries. Wastewater-based epidemiology (WBE) is potentially effective for early warning of a COVID-19 outbreak. In this study, presence of SARS-CoV-2 RNA in wastewater samples was investigated and was compared with the number of the confirmed COVID-19 cases in the study area during COVID-19 outbreak in Japan. In total, 45 influent wastewater samples were collected from five wastewater treatment plants in Ishikawa and Toyama prefectures in Japan. During the study period, the numbers of confirmed COVID-19 cases in these prefectures increased from 0.3 and 0 to >20 per 100,000 people. SARS-CoV-2 ribonucleic acid (RNA) in the samples was detected using several PCR-based assays. Of the 45 samples, 21 were positive for SARS-CoV-2 according to at least one of the three quantitative RT-PCR assays. The detection frequency increased when the number of total confirmed SARS-CoV-2 cases in 100,000 people exceeded 10 in each prefecture; however, SARS-CoV-2 could also be detected at a low frequency even when the number was below 1.0. SARS-CoV-2 in wastewater could be detected in the early stage of the epidemic, even if the number of confirmed cases potentially underestimates the actual numbers of cases. This suggests that WBE approach can potentially act as an early warning of COVID-19 outbreaks in Japan.