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Porous carbons are well-known efficient adsorbents for a variety of organic and inorganic pollutants, however, they have difficulty in virus removal. In this study, novel porous carbons (NPCs) (NPC-A, NPC-B, and NPC-C) derived from rice husks were compared with commercially available activated carbons (ACs) for their ability to remove MS2 bacteriophages (MS2) in a batch experiment. NPC-A was produced by the silica removal process. NPC-B was prepared with an additional steam activation applied to NPC-A. NPC-C was obtained with an additional acid rinse applied to NPC-B. The NPCs (particularly NPC-C) exhibited effective removal of up to 5.3 log10 of MS2, which was greater than that of less than 2.7 log10 obtained by other ACs under 10 g/L during the same contact time (60 min). The pore size distribution of the porous carbon adsorbents was found to influence their virus removal performance. The adsorbents with a larger proportion of pores ranging from 200–4500 nm in diameter were able to achieve higher virus removal rates. Thus, NPCs (particularly NPC-C), which had a larger volume of pores ranging from 200–4500 nm in size, demonstrated the potential for use as efficient adsorbents for removing viruses during water purification.

The presence of pathogenic viruses in drinking water is a major public health concern. Although viability RT-qPCR methods were developed to quantify infectious viruses, they may not always reflect viral infectivity, therefore leading to false-positive results. In this study, sodium deoxycholate (SD) pre-treatment was used to improve the efficiency of viability RT-qPCR methods with respect to exclusive quantification of infectious viruses. The ability of SD pre-treatment to enhance the penetration of three viability markers, namely, ethidium monoazide (EMA, 100 µM), propidium monoazide (PMA, 100 µM), and cis-dichlorodiammineplatinum (CDDP, 1000 µM), into heat-treated (90 °C for 1 min) Aichi virus at various concentrations (0.01–0.5%) was evaluated. The optimal SD concentration was found to be 0.1% for all markers. EMA/PMA/CDDP-RT-qPCR with 0.1% SD pre-treatment was significantly more effective than without SD pre-treatment in determining AiV inactivation after heat (50, 60, 70, 80, or 90 °C for 1 min) or chlorine treatment (1 mgCl2/L for 1, 2, 5, or 10 min). Among the viability RT-qPCR methods tested, CDDP-RT-qPCR with SD pre-treatment (SD-CDDP-RT-qPCR) was the most effective in reflecting viral infectivity. Performance testing of SD-CDDP-RT-qPCR in concentrated drinking water samples did not reveal any significant effects of SD-CDDP treatment. Thus, SD-CDDP-RT-qPCR could be a useful tool for monitoring infectious virus presence in drinking water.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes have been detected in wastewater worldwide. However, the assessment of SARS-CoV-2 infectivity in wastewater has been limited due to the stringent requirements of biosafety level 3. The main objective of this study is to investigate the applicability of capsid integrity RT-qPCR for the selective detection of intact SARS-CoV-2 in wastewater. Three capsid integrity reagents, namely ethidium monoazide (EMA, 0.1–100 μM), propidium monoazide (PMA, 0.1–100 μM), and cis-dichlorodiammineplatinum (CDDP, 0.1–1000 μM), were tested for their effects on different forms (including free genomes, intact and heat-inactivated) of murine hepatitis virus (MHV), which was used as a surrogate for SARS-CoV-2. CDDP at a concentration of 100 μM was identified as the most efficient reagent for the selective detection of infectious MHV by RT-qPCR (CDDP-RT-qPCR). Next, two common virus concentration methods including ultrafiltration (UF) and polyethylene glycol (PEG) precipitation were investigated for their compatibility with capsid integrity RT-qPCR. The UF method was more suitable than the PEG method since it recovered intact MHV (mean ± SD, 38% ± 29%) in wastewater much better than the PEG method did (0.013% ± 0.015%). Finally, CDDP-RT-qPCR was compared with RT-qPCR alone for the detection of SARS-CoV-2 in 16 raw wastewater samples collected in the Greater Tokyo Area. Five samples were positive for SARS-CoV-2 when evaluated by RT-qPCR alone. However, intact SARS-CoV-2 was detected in only three positive samples when determined by CDDP-RT-qPCR. Although CDDP-RT-qPCR was unable to determine the infectivity of SARS-CoV-2 in wastewater, this method could improve the interpretation of positive results of SARS-CoV-2 obtained by RT-qPCR., Graphical abstract Unlabelled Image

It is important to evaluate the removal of enteric viruses by drinking water treatment processes so that viral infection risk can be assessed and managed. However, evaluating the removal of enteric viruses by full-scale treatment processes can be challenging due to the low numbers of viruses and the presence of substances in the water samples that inhibit detection. In this study, we evaluated the removal of pepper mild mottle virus (PMMoV) by microfiltration (MF) and slow sand filtration (SSF) at two full-scale drinking water treatment plants in Japan, quantifying virus concentrations with real-time polymerase chain reaction (qPCR). The removal of PMMoV by MF ranged from 0.0 to >0.9 log10, although concentrations were below the detection limit for half of the treated water samples. SSF removed PMMoV by up to 2.8 log10; however, the removal efficiency decreased to 0.0–1.0 log10 under cold water temperatures. Process control showed that nucleic acid extraction and qPCR efficiency were inhibited in nearly 40% of water samples. Dilution, DAX-8, and ferrihydrite treatments for purification were effective in mitigating these inhibitory effects.

Capsid integrity (RT-)qPCR has recently been developed to discriminate between intact forms from inactivated forms of viruses, but its applicability to identifying integrity of viruses in drinking water has remained limited. In this study, we investigated the application of capsid integrity (RT-)qPCR using cis-dichlorodiammineplatinum (CDDP) with sodium deoxycholate (SD) pretreatment (SD-CDDP-(RT-)qPCR) to detect intact viruses in surface water and tap water. A total of 63 water samples (surface water, n = 20; tap water, n = 43) were collected in the Kanto region in Japan and quantified by conventional (RT)-qPCR and SD-CDDP-(RT-)qPCR for pepper mild mottle virus (PMMoV) and seven other viruses pathogenic to humans (Aichivirus (AiV), noroviruses of genotypes I and II, enterovirus, adenovirus type 40 and 41, and JC and BK polyomaviruses). In surface water, PMMoV (100%) was more frequently detected than other human pathogenic viruses (30%–60%), as determined by conventional (RT-)qPCR. SD-CDDP-(RT-)qPCR also revealed that intact PMMoV (95%) was more common than intact human pathogenic viruses (20%–45%). In the tap water samples, most of the target viruses were not detected by conventional (RT-)qPCR, except for PMMoV (9%) and AiV (5%). PMMoV remained positive (5%), whereas no AiV was detected when tested by SD-CDDP-(RT-)qPCR, indicating that some PMMoV had an intact capsid, whereas AiV had damaged capsids. The presence of AiV in the absence of PMMoV in tap water produced from groundwater may demonstrate the limitation of PMMoV as a viral indicator in groundwater. In addition to being abundant in surface water, PMMoV was detected in tap water, including PMMoV with intact capsids. Thus, the absence of intact PMMoV may be used to guarantee the viral safety of tap water produced from surface water.

Molecular assays are currently the most widely used method to quantify pathogenic viruses in water; however, their performance is often disrupted by matrices present in environmental samples. The present study used ferrihydrite (Fh) treatment to mitigate inhibition of RT-qPCR virus detection from environmental water concentrates. Fh treatment was performed to improve the detection of spiked Aichi virus 1 (AiV) and Qβ bacteriophage (Qβ) in commercial humic acids and seawater concentrates. The optimal Fh doses were found to be 200, 500, and 1000 mgFe/L for humic acid concentrates at UV254 of 1.5, 3.0, and 6.0 cm-1 respectively, whereas this value was 1000 mgFe/L for the seawater concentrates at UV254 of 1.5 cm-1. At these optimal doses, the recoveries of spiked AiV and Qβ ranged from 2% to 12% in the humic acid and seawater concentrates, respectively. In addition, high levels of indigenous viruses (including AiV ≥ 2.81 log10 copies/mL and pepper mild mottle virus ≥ 2.77 log10 copies/mL) were detected from seawater concentrates after Fh treatment, while none were detected without Fh treatment. Fh treatment effectively mitigated the inhibitive effects from environmental water samples.