Evaluation of reduction efficiencies of pepper mild mottle virus and human enteric viruses in full-scale drinking water treatment plants employing coagulation-sedimentation-rapid sand filtration or coagulation-microfiltration.

Here, we evaluated the reduction efficiencies of indigenous pepper mild mottle virus (PMMoV, a potential surrogate for human enteric viruses to assess virus removal by coagulation-sedimentation-rapid sand filtration [CS-RSF] and coagulation-microfiltration [C-MF]) and representative human enteric viruses in four full-scale drinking water treatment plants that use CS-RSF (Plants A and B) or C-MF (Plants C and D). First, we developed a virus concentration method by using an electropositive filter and a tangential-flow ultrafiltration membrane to effectively concentrate and recover PMMoV from large volumes of water: the recovery rates of PMMoV were 100% when 100-L samples of PMMoV-spiked dechlorinated tap water were concentrated to 20 mL; even when spiked water volume was 2000 L, recovery rates of >30% were maintained. The concentrations of indigenous PMMoV in raw and treated water samples determined by using this method were always above the quantification limit of the real-time polymerase chain reaction assay. We therefore were able to determine its reduction ratios: 0.9-2.7-log10 in full-scale CS-RSF and 0.7-2.9-log10 in full-scale C-MF. The PMMoV reduction ratios in C-MF at Plant C (1.0 ± 0.3-log10) were lower than those in CS-RSF at Plants A (1.7 ± 0.5-log10) and B (1.4 ± 0.7-log10), despite the higher ability of MF for particle separation in comparison with RSF owing to the small pore size in MF. Lab-scale virus-spiking C-MF experiments that mimicked full-scale C-MF revealed that a low dosage of coagulant (polyaluminum chloride [PACl]) applied in C-MF, which is determined mainly from the viewpoint of preventing membrane fouling, probably led to the low reduction ratios of PMMoV in C-MF. This implies that high virus reduction ratios (>4-log10) achieved in previous lab-scale virus-spiking C-MF studies are not necessarily achieved in full-scale C-MF. The PMMoV reduction ratios in C-MF at Plant D (2.2 ± 0.6-log10) were higher than those at Plant C, despite similar coagulant dosages. In lab-scale C-MF, the PMMoV reduction ratios increased from 1-log10 (with PACl [basicity 1.5], as at Plant C) to 2-4-log10 (with high-basicity PACl [basicity 2.1], as at Plant D), suggesting that the use of high-basicity PACl probably resulted in higher reduction ratios of PMMoV at Plant D than at Plant C. Finally, we compared the reduction ratios of indigenous PMMoV and representative human enteric viruses in full-scale CS-RSF and C-MF. At Plant D, the concentrations of human norovirus genogroup II (HuNoV GII) in raw water were sometimes above the quantification limit; however, whether its reduction ratios in C-MF were higher than those of PMMoV could not be judged since reduction ratios were >1.4-log10 for HuNoV GII and 2.3-2.9-log10 for PMMoV. At Plant B, the concentrations of enteroviruses (EVs) and HuNoV GII in raw water were above the quantification limit on one occasion, and the reduction ratios of EVs (>1.2-log10) and HuNoV GII (>1.5-log10) in CS-RSF were higher than that of PMMoV (0.9-log10). This finding supports the usefulness of PMMoV as a potential surrogate for human enteric viruses to assess virus removal by CS-RSF.

Suitability of pepper mild mottle virus as a human enteric virus surrogate for assessing the efficacy of thermal or free-chlorine disinfection processes by using infectivity assays and enhanced viability PCR.

Evaluating the efficacy of disinfection processes to inactivate human enteric viruses is important for the prevention and control of waterborne diseases caused by exposure to those viruses via drinking water. Here, we evaluated the inactivation of two representative human enteric viruses (adenovirus type 40 [AdV] and coxsackievirus B5 [CV]) by thermal or free-chlorine disinfection. In addition, we compared the infectivity reduction ratio of a plant virus (pepper mild mottle virus [PMMoV], a recently proposed novel surrogate for human enteric viruses for the assessment of virus removal by coagulation‒rapid sand filtration and membrane filtration) with that of the two human enteric viruses to assess the suitability of PMMoV as a human enteric virus surrogate for use in thermal and free-chlorine disinfection processes. Finally, we examined whether conventional or enhanced viability polymerase chain reaction (PCR) analysis using propidium monoazide (PMA) or improved PMA (PMAxx) with or without an enhancer could be used as alternatives to infectivity assays (i.e., plaque-forming unit method for AdV and CV; local lesion count assay for PMMoV) for evaluating virus inactivation by disinfection processes. We found that PMMoV was more resistant to heat treatment than AdV and CV, suggesting that PMMoV is a potential surrogate for these two enteric viruses with regard to thermal disinfection processes. However, PMMoV was much more resistant to chlorine treatment compared with AdV and CV (which is chlorine-resistant) (CT value for 4-log10 inactivation: PMMoV, 84.5 mg-Cl2·min/L; CV, 1.15-1.19 mg-Cl2·min/L), suggesting that PMMoV is not useful as a surrogate for these enteric viruses with regard to free-chlorine disinfection processes. For thermal disinfection, the magnitude of the signal reduction observed with PMAxx-Enhancer-PCR was comparable with the magnitude of reduction in infectivity, indicating that PMAxx-Enhancer-PCR is a potential alternative to infectivity assay. However, for free-chlorine disinfection, the magnitude of the signal reduction observed with PMAxx-Enhancer-PCR was smaller than the magnitude of the reduction in infectivity, indicating that PMAxx-Enhancer-PCR underestimated the efficacy of virus inactivation (i.e., overestimated the infectious virus concentration) by chlorine treatment. Nevertheless, among the PCR approaches examined in the present study (PCR alone, PMA-PCR or PMAxx-PCR either with or without enhancer), PMAxx-Enhancer-PCR provided the most accurate assessment of the efficacy of virus inactivation by thermal or free chlorine disinfection processes.