The primary concentration and molecular process are critical to implement wastewater-based epidemiology for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the previously developed methods were optimized for nonenveloped viruses. Few studies evaluated if the methods are applicable to the efficient recovery of enveloped viruses from various types of raw sewage. This study aims (1) to compare the whole process recovery of Pseudomonas phage φ6, a surrogate for enveloped viruses, among combinations of primary concentration [ultrafiltration (UF), electronegative membrane vortex (EMV), and polyethylene glycol precipitation (PEG)] and RNA extraction methods (spin column-based method using QIAamp Viral RNA Mini Kit and acid guanidinium thiocyanate–phenol–chloroform extraction using TRIzol reagent) for three types of raw sewage and (2) to test the applicability of the method providing the highest φ6 recovery to the detection of SARS-CoV-2 RNA. Among the tested combinations, PEG+TRIzol provided the highest φ6 recovery ratio of 29.8% to 49.8% (geometric mean). UF+QIAamp Viral RNA Mini Kit provided the second highest φ6 recovery of 6.4% to 35.8%. The comparable φ6 recovery was observed for UF+TRIzol (13.8 – 30.0 %). PEG+QIAamp Viral RNA Mini Kit provided only 1.4% to 3.0% of φ6 recovery, while coliphage MS2, a surrogate for nonenveloped viruses, was recovered comparably with PEG+TRIzol. This indicated that the nonenveloped surrogate (MS2) did not necessarily validate the efficient recovery for enveloped viruses. EMV+QIAamp Viral RNA Mini Kit provided significantly different φ6 recovery (1.6 – 21 %) among the types of raw sewage. Then, the applicability of modified PEG+TRIzol was examined for the raw sewage collected in Tokyo, Japan. Of the 12 grab samples, 4 were positive for SARS-CoV-2 CDC N1 and N3 assay. Consequently, PEG+TRIzol provided the highest φ6 recovery and allowed for the detection of SARS-CoV-2 RNA from raw sewage.

Here, we describe a strategy to obtain nanoporous liquid-crystalline (LC) membranes by incorporating a photocleavable ortho-nitrobenzyl group in polymerizable columnar liquid crystals. Two derivatives were synthesized with propylene and nonylene spacers, respectively, between the ionic and the photocleavable moieties to introduce various size nanopores after photocleavage. The membranes were prepared by photopolymerization in the LC states, followed by photocleavage and washing with methanol. The resulting membranes show a virus rejection of 99.99%. Although the rejection value remained almost the same for the two membranes, water flux increased with increasing the length of the alkyl spacers. These membranes were found to be almost free from pinhole defects. The present study offers a new methodology for the development of nanoporous membranes with organized nanostructures for separation technologies.

We have developed a two-dimensional (2D) liquidcrystalline (LC) nanostructured water-treatment membrane showing high virus rejection ability (over 99.99997% for bacteriophage Qβ) and improved water permeation. Polymerizable gemini amphiphiles have been designed and synthesized. They have H-shaped gemini-type structures of thermotropic smectic liquid crystals composed of cationic imidazolium moieties. One of the gemini amphiphiles shows a smectic A phase with an interdigitated bilayer structure. A cross-linked self-standing 2D nanostructured polymer film has been obtained by in situ photopolymerization of the gemini amphiphile in the smectic phase. The length of linkers in gemini amphiphiles affects the formation of LC phases. The 2D nanostructured membrane also showed selective salt rejection.

The disinfection susceptibilities of viruses vary even among variants, yet the inactivation efficiency of a certain virus genotype, species, or genus was determined based on the susceptibility of its laboratory strain. The objectives were to evaluate the variability in susceptibilities to free chlorine, UV254, and ozone among 13 variants of coxsackievirus B5 (CVB5) and develop the model allowing for predicting the overall inactivation of heterogeneous CVB5. Our results showed that the susceptibilities differed by up to 3.4-fold, 1.3-fold, and 1.8-fold in free chlorine, UV254, and ozone, respectively. CVB5 in genogroup B exhibited significantly lower susceptibility to free chlorine and ozone than genogroup A, where the laboratory strain, Faulkner, belongs. The capsid protein in genogroup B contained a lower number of sulfur-containing amino acids, readily reactive to oxidants. We reformulated the Chick–Watson model by incorporating the probability distributions of inactivation rate constants to capture the heterogeneity. This expanded Chick–Watson model indicated that up to 4.2-fold larger free chlorine CT is required to achieve 6-log inactivation of CVB5 than the prediction by the Faulkner strain. Therefore, it is recommended to incorporate the variation in disinfection susceptibilities for predicting the overall inactivation of a certain type of viruses.

Polyethylene glycol (PEG) precipitation is one of the conventional methods for virus concentration. This technique has been used to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in wastewater. The procedures and seeded surrogate viruses were different among implementers; thus, the reported whole process recovery efficiencies considerably varied among studies. The present study compared five PEG precipitation procedures, with different operational parameters, for the RT-qPCR-based whole process recovery of murine hepatitis virus (MHV), bacteriophage phi6, and pepper mild mottle virus (PMMoV), and molecular process recovery of murine norovirus using 34 raw wastewater samples collected in Japan. The five procedures yielded significantly different whole process recovery of MHV (0.070%–2.6%) and phi6 (0.078%–0.51%). The observed concentration of indigenous PMMoV ranged from 8.9 to 9.7 log (7.9 × 10 8 to 5.5 × 10 9 ) copies/L. Interestingly, PEG precipitation with 2-h incubation outperformed that with overnight incubation partially due to the difference in molecular process recovery efficiency. The recovery load of MHV exhibited a positive correlation ( r = 0.70) with that of PMMoV, suggesting that PMMoV is the potential indicator of the recovery efficiency of SARS-CoV-2. In addition, we reviewed 13 published studies and found considerable variability between different studies in the whole process recovery efficiency of enveloped viruses by PEG precipitation. This was due to the differences in operational parameters and surrogate viruses as well as the differences in wastewater quality and bias in the measurement of the seeded load of surrogate viruses, resulting from the use of different analytes and RNA extraction methods. Overall, the operational parameters (e.g., incubation time and pretreatment) should be optimized for PEG precipitation. Co-quantification of PMMoV may allow for the normalization of SARS-CoV-2 RNA concentration by correcting for the differences in whole process recovery efficiency and fecal load among samples.

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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.