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Viruses can interact with a broad range of inorganic and organic particles in water and wastewater. These associations can protect viruses from inactivation by quenching chemical disinfectants or blocking ultraviolet light transmission, and a much higher dosage of disinfectants is required to inactivate particle-associated viruses than free viruses. There have been only few studies of the association of viruses with particles in wastewater, particularly in secondary treated effluent. As secondary effluent is the source water to the reclaimed water treatment system, this study quantified indigenous enteric viruses, and viral indicators associated with particles in secondary effluents collected from five full-scale water reclamation facilities in the United States. Particle-associated viruses were enumerated using a sequential filtration followed by microfluidic digital PCR. This study showed that enteric viruses and viral indicators (crAssphage and pepper mild mottle virus, PMMoV) were attached to particles of different sizes in secondary effluent. Significantly higher concentrations of RNA viruses including PMMoV, norovirus, and enterovirus were detected in filtrate of the sequential filtration, which contained particles < 0.45 µm. DNA viruses including adenovirus and crAssphage were found to be more associated with larger particles in secondary effluent. Overall, high correlations were observed between viral indicators and enteric viruses, supporting the use of crAssphage and PMMoV to evaluate virus removal efficiency in water and wastewater treatment processes. The association of viruses with particles in wastewater has significant implications on wastewater treatment and disinfection processes as well as virus enumeration in wastewater. [ABSTRACT FROM AUTHOR]

Pathogenic viruses in environmental water are usually present in levels too low for direct detection and thus, a concentration step is often required to increase the analytical sensitivity. The objective of this study was to evaluate an automated filtration device, the Innovaprep Concentrating Pipette Select (CP Select) for the rapid concentration of viruses in saline water samples, while considering duration of process and ease of use. Four bacteriophages (MS2, P22, Phi6, and PhiX174) and three animal viruses (adenovirus, coronavirus OC43, and canine distemper virus) were seeded in artificial seawater, aquarium water, and bay water samples, and processed using the CP Select. The recovery efficiencies of viruses were determined either using a plaque assay or droplet digital PCR (ddPCR). Using plaque assays, the average recovery efficiencies for bacteriophages ranged from 4.84 ± 3.8% to 82.73 ± 27.3%, with highest recovery for P22 phage. The average recovery efficiencies for the CP Select were 39.31 ± 26.6% for adenovirus, 19.04 ± 11.6% for coronavirus OC43, and 19.84 ± 13.6% for canine distemper virus, as determined by ddPCR. Overall, viral genome composition, not the size of the virus, affected the recovery efficiencies for the CP Select. The small sample volume size used for the ultrafilter pipette of the system hinders the use of this method as a primary concentration step for viruses in marine waters. However, the ease of use and rapid processing time of the CP Select are especially beneficial when rapid detection of viruses in highly contaminated water, such as wastewater or sewage-polluted surface water, is needed. [ABSTRACT FROM AUTHOR]

Recent studies have shown that Lake Pontchartrain Estuary in Louisiana experiences frequent harmful cyanobacterial blooms (cyanoHABs). In 2019, the Bonnet Carré Spillway (BCS) that diverts Mississippi River water into the estuary opened twice in the same year for the first time in history to prevent flooding in New Orleans. Short-term water quality monitoring was conducted in shoreline areas with high public use for the presence of cyanoHABs and cyanotoxins to assess the public health risks. Field sampling methods and satellite imagery were used to determine water quality and quantify bloom intensity and toxicity across time and space. Long-term BCS opening created a fresh (salinity < 0.2) and nutrient-rich estuary that supported several cyanoHABs in warmer months during and after the second BCS closure. Cyanobacterial biomass ranged from 35 to 4972 µg PC L−1, while toxin microcystin ranged from undetected to 8.41 µg MC L−1. The highest biomass and toxin were detected on June 25 at the north shore, station LP8, Mandeville Beach, dominated by Microcystis and Dolichospermum species. CyanoHABs occurred mostly in the northern part of the estuary, where tributary discharge is also a strong influence. Some of these blooms exited the estuary and were transported to the Gulf of Mexico following passage through Lake Borgne and then Mississippi Sound. Modifications in the timing and duration of river diversion operations can create prolonged cyanobacterial blooms that can cause environmental and public health risks, especially in warmer months, and this may intensify due to a changing climate. [ABSTRACT FROM AUTHOR]

Wastewater surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emerging public health tool to understand the spread of Coronavirus Disease 2019 (COVID-19) in communities. The performance of different virus concentration methods and PCR methods needs to be evaluated to ascertain their suitability for use in the detection of SARS-CoV-2 in wastewater. We evaluated ultrafiltration and polyethylene glycol (PEG) precipitation methods to concentrate SARS-CoV-2 from sewage in wastewater treatment plants and upstream in the wastewater network (e.g., manholes, lift stations). Recovery of viruses by different concentration methods was determined using Phi6 bacteriophage as a surrogate for enveloped viruses. Additionally, the presence of SARS-CoV-2 in all wastewater samples was determined using reverse transcription quantitative PCR (RT-qPCR) and reverse transcription droplet digital PCR (RT-ddPCR), targeting three genetic markers (N1, N2 and E). Using spiked samples, the Phi6 recoveries were estimated at 2.6–11.6% using ultrafiltration-based methods and 22.2–51.5% using PEG precipitation. There was no significant difference in recovery efficiencies (p < 0.05) between the PEG procedure with and without a 16 h overnight incubation, demonstrating the feasibility of obtaining same day results. The SARS-CoV-2 genetic markers were more often detected by RT-ddPCR than RT-qPCR with higher sensitivity and precision. While all three SARS-CoV-2 genetic markers were detected using RT-ddPCR, the levels of E gene were almost below the limit of detection using RT-qPCR. Collectively, our study suggested PEG precipitation is an effective low-cost procedure which allows a large number of samples to be processed simultaneously in a routine wastewater monitoring for SARS-CoV-2. RT-ddPCR can be implemented for the absolute quantification of SARS-CoV-2 genetic markers in different wastewater matrices. [ABSTRACT FROM AUTHOR]

The quantification and trends in concentrations for naturally occurring rotaviruses (RV) and enteroviruses (EV) in untreated sewage in various wastewater systems have not often been compared. There is now greater interest in monitoring the infections in the community including live vaccine efficacy by evaluating untreated sewage. The goals of this study were to 1) survey the concentrations of naturally occurring RV and EV in untreated sewage using a reverse transcription—droplet digital polymerase chain reaction (RT-ddPCR) and 2) investigate the use of a new adsorption elution (bag-mediated filtration system (BMFS) using ViroCap filters) against more traditional polyethylene glycol (PEG) precipitation for virus concentration. Sewage samples were collected from lagoons in Kenya and Michigan (MI), the United States (USA) and from wastewater treatment plants (WWTPs) in the USA. RVs were detected at geometric mean concentrations in various locations, California (CA) 1.31 × 105 genome copies/L (gc/L), Kenya (KE) 2.71 × 104 gc/L and Virginia (VA) 1.48 × 105 gc/L, and EVs geometric means were 3.72 × 106 gc/L (CA), 1.18 × 104 gc/L (Kenya), and 6.18 × 103 gc/L (VA). The mean RV concentrations using BMFS-ViroCap in split samples compared to PEG precipitation methods demonstrated that the levels were only 9% (#s BMFS/PEG) in the Michigan lagoons which was significantly different (p < 0.01). This suggests that RV concentrations in Kenya are around 1.69 × 106 gc/L. Overall, there was no difference in concentrations for the other sampling locations across the methods of virus recovery (i.e., PEG precipitation and HA filters) using one-way ANOVA (F = 1.7, p = 0.2739) or Tukey–Kramer pairwise comparisons (p > 0.05). This study provides useful data on RV and EV concentrations in untreated sewage in Kenya and the USA. It also highlights on the usefulness of the RT-ddPCR for absolute quantification of RV and EV in sewage samples. The BMFS using ViroCap filters while less efficient compared to the more traditional PEG precipitation method was able to recover RVs and EVs in untreated sewage and may be useful in poor resource settings while underestimating viruses by 1 to 1.5 logs. [ABSTRACT FROM AUTHOR]