Implementation of a Sensitive Method to Assess High Virus Retention Performance of Low-Pressure Reverse Osmosis Process.

Human enteric viruses are important etiological agents of waterborne diseases. Environmental waters are usually contaminated with low virus concentration requiring large concentration factors for effective detection by (RT)-qPCR. Low-pressure reverse osmosis is often used to remove water contaminants, but very few studies focused on the effective virus removal of reverse osmosis treatment with feed concentrations as close as possible to environmental concentrations and principally relied on theoretical virus removal. The very low viral concentrations usually reported in the permeates (i.e. at least 5 log of removal rate) mean that very large volumes of water need to be analysed to have sufficient sensitivity and assess the process efficiency. This study evaluates two methods for the concentration of adenoviruses, enteroviruses and MS2 bacteriophages at different viral concentrations in large (< 200 L) and very large (> 200 L) volumes. The first method is composed of two ultrafiltration membranes with low-molecular weight cut-offs while the second method primarily relies on adsorption and elution phases using electropositive-charged filters. The recovery rates were assessed for both methods. For the ultrafiltration-based protocol, recovery rates were similar for each virus studied: 80% on average at high virus concentrations (106-107 viruses L-1) and 50% at low virus concentrations (103-104 viruses L-1). For the electropositive-charged filter-based method, the average recoveries obtained were about 36% for ADV 41, 57% for CV-B5 and 1.6% for MS2. The ultrafiltration-based method was then used to evaluate the performance of a low-pressure reverse osmosis lab-scale pilot plant. The retentions by reverse osmosis were similar for all studied viruses and the validated recovery rates applied to the system confirmed the reliability of the concentration method. This method was effective in concentrating all three viruses over a wide range of viral concentrations. Moreover, the second concentration method using electropositive-charged filters was studied, allowing the filtration of larger volumes of permeate from a semi-industrial low-pressure reverse osmosis pilot plant. This reference method was used because of the inability of the UF method to filter volumes on the order of one cubic metre.

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.

A novel approach to concentrate human and animal viruses from wastewater using receptors-conjugated magnetic beads.

Viruses are present at low concentrations in wastewater; therefore, an effective method for concentrating virus particles is necessary for accurate wastewater-based epidemiology (WBE). We designed a novel approach to concentrate human and animal viruses from wastewater using porcine gastric mucin-conjugated magnetic beads (PGM-MBs). We systematically evaluated the performances of the PGM-MBs method (sensitivity, specificity, and robustness to environmental inhibitors) with six viral species, including Tulane virus (a surrogate for human norovirus), rotavirus, adenovirus, porcine coronavirus (transmissible gastroenteritis virus or TGEV), and two human coronaviruses (NL63 and SARS-CoV-2) in influent wastewater and raw sewage samples. We determined the multiplication factor (the ratio of genome concentration of the final solution to that of the initial solution) for the PGM-MBs method, which ranged from 1.3 to 64.0 depending on the viral species. Because the recovery efficiency was significantly higher when calculated with virus titers than it was with genome concentration, the PGM-MBs method could be an appropriate tool for assessing the risk to humans who are inadvertently exposed to wastewater contaminated with infectious viruses. Furthermore, PCR inhibitors were not concentrated by PGM-MBs, suggesting that this tool will be successful for use with environmental samples. In addition, the PGM-MBs method is cost-effective (0.5 USD/sample) and has a fast turnaround time (3 h from virus concentration to genome quantification). Thus, this method can be implemented in high throughput facilities. Because of its strong performance, intrinsic characteristics of targeting the infectious virus, robustness to wastewater, and adaptability to high throughput systems, the PGM-MBs method can be successfully applied to WBE and ultimately provides valuable public health information.

A review on detection of SARS-CoV-2 RNA in wastewater in light of the current knowledge of treatment process for removal of viral fragments.

The entire globe is affected by the novel disease of coronavirus 2019 (COVID-19 or 2019-nCoV), which is formally recognised as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The World Health Organisation (WHO) announced this disease as a global pandemic. The presence of SARS-CoV-2 RNA in unprocessed wastewater has become a cause of worry due to these emerging pathogens in the process of wastewater treatment, as reported in the present study. This analysis intends to interpret the fate, environmental factors and route of transmission of SARS-CoV-2, along with its eradication by treating the wastewater for controlling and preventing its further spread. Different recovery estimations of the virus have been depicted by the detection of SARS-CoV-2 RNA in wastewater through the viral concentration techniques. Most frequently used viral concentration techniques include polyethylene glycol (PEG) precipitation, ultrafiltration, electronegative membrane, and ultracentrifugation, after which the detection and quantification of SARS-CoV-2 RNA are done in wastewater samples through quantitative reverse transcription-polymerase chain reaction (RT-qPCR). The wastewater treatment plant (WWTP) holds the key responsibility of eliminating pathogens prior to the discharge of wastewater into surface water bodies. The removal of SARS-CoV-2 RNA at the treatment stage is dependent on the operations of wastewater treatment systems during the outbreak of the virus; particularly, in the urban and extensively populated regions. Efficient primary, secondary and tertiary methods of wastewater treatment and disinfection can reduce or inactivate SARS-CoV-2 RNA before being drained out. Nonetheless, further studies regarding COVID-19-related disinfectants, environment conditions and viral concentrations in each treatment procedure, implications on the environment and regular monitoring of transmission need to be done urgently. Hence, monitoring the SARS-CoV-2 RNA in samples of wastewater under the procedure of wastewater-based epidemiology (WBE) supplement the real-time data pertaining to the investigation of the COVID-19 pandemic in the community, regional and national levels.

Benchmarking virus concentration methods for quantification of SARS-CoV-2 in raw wastewater.

Wastewater-based epidemiology offers a cost-effective alternative to testing large populations for SARS-CoV-2 virus, and may potentially be used as an early warning system for SARS-CoV-2 pandemic spread. However, viruses are highly diluted in wastewater, and a validated method for their concentration and further processing, and suitable reference viruses, are the main needs to be established for reliable SARS-CoV-2 municipal wastewater detection. For this purpose, we collected wastewater from two European cities during the Covid-19 pandemic and evaluated the sensitivity of RT-qPCR detection of viral RNA after four concentration methods (two variants of ultrafiltration-based method and two adsorption and extraction-based methods). Further, we evaluated one external (bovine corona virus) and one internal (pepper mild mottle virus) reference virus. We found a consistently higher recovery of spiked virus using the modified ultrafiltration-based method. This method also had a significantly higher efficiency (p-value <0.01) for wastewater SARS-CoV-2 detection. The ultracentrifugation method was the only method that detected SARS-CoV-2 in the wastewater of both cities. The pepper mild mottle virus was found to function as a potentially suitable internal reference standard.

A comparison of virus concentration methods for molecular detection and characterization of rotavirus in bivalve shellfish species.

The objectives of this study were to develop a method for concentrating rotavirus, to assess the detection rate, and to characterize the genotype of naturally occurring rotavirus in bivalve shellfish species; including oysters (Saccostrea forskali), cockles (Anadara nodifera), and mussels (Perna viridis). The results demonstrated that an adsorption-twice elution-extraction method was less-time consuming method of concentrating the spiked rotavirus, yielding high sensitivity of 1.14 genome copies/g of digestive tissues from all three shellfish species, as detected using an RT-nested PCR. In seeding experiments, rotavirus as low as 1.39 genome copies was able to be detected in 4 g of digestive tissues or per sample. In the period of August 2011 to July 2012, of the 300 bivalve shellfish samples collected and tested, 24 (8.0%) were found to be contaminated with rotavirus, the figures being: oysters, 13/100 samples; mussels, 10/100 samples; and cockles, 1/100 samples. By DNA sequencing of the RT-nested PCR products and phylogenetic analysis, the rotaviruses detected were classified into G1, lineage II (4 samples); G3 (10 samples): lineage I (3 samples), lineage IIIc (3 samples), lineage IIId (3 samples), lineage IV (1 sample); G9 (6 samples); and G12, lineage III (1 sample). These findings suggest that this virus concentration method provides high sensitivity for the detection of rotavirus from the three bivalve shellfish species. The prevalence of rotavirus and the identified genotypes contribute to the molecular epidemiology of rotavirus in different shellfish species.

A simple and efficient method for detecting avian influenza virus in water samples.

Waterborne transmission plays an essential role in the transmission and spread of avian influenza viruses. The abundance of influenza viruses in environmental water is usually extremely low and viruses or viral genomes can hardly be detected by conventional reverse transcription (RT-) PCR without concentration. In the present study, an electropositive filter membrane was used to concentrate influenza viruses from water sample, in addition, a glass fiber filter has been used prior to positive charged membrane for the prefiltration. Unlike the traditional adsorption-elution method, Trizol-LS reagent was used to lyse the viruses attached directly to the electropositive filter membrane and the influenza virus genomic RNA was extracted, followed by RT-PCR analysis. The method established in this study could improve the efficiency of the conventional RT-PCR technique used to detect the M, NP, and HA genes of influenza virus in natural water samples. This method could also reduce the time taken for the traditional adsorption-elution concentration procedure.