Genetically distinct genogroup IV norovirus strains identified in wastewater.

We investigated the prevalence and genetic diversity of genogroup IV norovirus (GIV NoV) strains in wastewater in Arizona, United States, over a 13-month period. Among 50 wastewater samples tested, GIV NoVs were identified in 13 (26 %) of the samples. A total of 47 different GIV NoV strains were identified, which were classified into two genetically distinct clusters: the GIV.1 human cluster and a unique genetic cluster closely related to strains previously identified in Japanese wastewater. The results provide additional evidence of the considerable genetic diversity among GIV NoV strains through the analysis of wastewater containing virus strains shed from all populations.

Occurrence and genetic diversity of human cosavirus in influent and effluent of wastewater treatment plants in Arizona, United States.

Human cosavirus (HCoSV) is a novel member of the family Picornaviridae. We investigated the prevalence and genetic diversity of HCoSV in influent and effluent wastewater in Arizona over a 12-month period, from August 2011 to July 2012. HCoSV sequences were identified in six (25%) influent samples and one (4%) effluent sample, with the highest concentration of 3.24 × 10(5) and 1.54 × 10(3) copies/liter in influent and effluent, respectively. The strains were characterized based on their 5' untranslated region and classified into species A and D, demonstrating that genetically heterogeneous HCoSV were circulating with a clear temporal shift of predominant strains in the study area.

Persistence of Infectious Human Norovirus in Estuarine Water.

Norovirus is the predominant cause of viral acute gastroenteritis globally. While person-to-person is the most reported transmission route, norovirus is also associated with waterborne and foodborne illness, including from the consumption of contaminated bivalve molluscan shellfish. The main cause of shellfish contamination is via the bioaccumulation of norovirus from growing waters impacted by human wastewater. However, data on the persistence of infectious norovirus in the environment are limited due to a lack of a human norovirus culture method in the past. In this study, we applied the recently established method of norovirus replication in human intestinal enteroids to determine the persistence of norovirus in artificial estuarine water at 25 ppt for up to 21 days at 4 °C and 16 °C in the dark. Infectious norovirus was detected for up to 21 days. The relative infectivity declined from 100 to 3% at day 21, with decay rate constants of 0.07 day-1 at 4 °C and 0.17 day-1 at 16 °C. There was no decrease in norovirus titres as measured by reverse transcription-droplet digital PCR (RT-ddPCR), confirming the lack of the relationship between norovirus infectivity and direct detection by PCR. The results confirm that norovirus can remain infectious for at least 3 weeks in an estuarine water environment, presenting associated health risks.

Public health use and lessons learned from a statewide SARS-CoV-2 wastewater monitoring program (MiNET).

The global SARS-CoV-2 monitoring effort has been extensive, resulting in many states and countries establishing wastewater-based epidemiology programs to address the spread of the virus during the pandemic. Challenges for programs include concurrently optimizing methods, training new laboratories, and implementing successful surveillance programs that can rapidly translate results for public health, and policy making. Surveillance in Michigan early in the pandemic in 2020 highlights the importance of quality-controlled data and explores correlations with wastewater and clinical case data aggregated at the state level. The lessons learned and potential measures to improve public utilization of results are discussed. The Michigan Network for Environmental Health and Technology (MiNET) established a network of laboratories that partnered with local health departments, universities, wastewater treatment plants (WWTPs) and other stakeholders to monitor SARS-CoV-2 in wastewater at 214 sites in Michigan. MiNET consisted of nineteen laboratories, twenty-nine local health departments, 6 Native American tribes, and 60 WWTPs monitoring sites representing 45 % of Michigan's population from April 6 and December 29, 2020. Three result datasets were created based on quality control criteria. Wastewater results that met all quality assurance criteria (Dataset Mp) produced strongest correlations with reported clinical cases at 16 days lag (rho = 0.866, p < 0.05). The project demonstrated the ability to successfully track SARS-CoV-2 on a large, state-wide scale, particularly data that met the outlined quality criteria and provided an early warning of increasing COVID-19 cases. MiNET is currently poised to leverage its competency to complement public health surveillance networks through environmental monitoring for new and emerging pathogens of concern and provides a valuable resource to state and federal agencies to support future responses.

Removal of fecal indicator bacteria and antibiotic resistant genes in constructed wetlands.

Wastewater discharge evidently increased bacterial diversity in the receiving waterbodies. The objective of this study was to evaluate the effectiveness of a constructed wetland in reducing fecal indicator bacteria (FIB) and antibiotic resistant genes (ARGs). We determined the prevalence and attenuation of fecal indicator bacteria including Escherichia coli and enterococci, along with ARGs, and human-associated Bacteroidales (HF183) markers by quantitative polymerase chain reaction (qPCR) method. Three types of water samples (inlet, intermediate, and outlet) from a constructed wetland were collected once a month from May to December in 2013. The overall reduction of E. coli was 50.0% based on culture method. According to the qPCR result, the overall removal rate of E. coli was only 6.7%. Enterococci were found in 62.5% of the wetland samples. HF183 genetic marker was detected in all final effluent samples with concentration ranging from 1.8 to 4.22 log10 gene copies (GC)/100 ml. Of the ARGs tested, erythromycin resistance genes (ermF) were detected in 79.2% of the wetland samples. The class 1 integrase (intI1) was detected in all water samples with concentration ranging from 0.83 to 5.54 log10 GC/100 ml. The overall removal rates of enterococci, HF183, intI1, and ermF were 84.0%, 66.6%, 67.2%, and 13.1%, respectively.

Human polyomavirus: Advantages and limitations as a human-specific viral marker in aquatic environments.

Human polyomaviruses (HPyVs) cause persistent infections in organs such as kidney, brain, skin, liver, respiratory tract, etc., and some types of HPyV are constantly excreted in the urine and/or feces of infected and healthy individuals. The use of an enteric virus as an indicator for human sewage/waste contamination in aquatic environments has been proposed; HPyVs are a good candidate since they are routinely found in environmental water samples from different geographical areas with relatively high abundance. HPyVs are highly human specific, having been detected in human waste from all age ranges and undetected in animal waste samples. In addition, HPyVs show a certain degree of resistance to high temperature, chlorine, UV, and low pH, with molecular signals (i.e., DNA) persisting in water for several months. Recently, various concentration methods (electronegative/positive filtration, ultrafiltration, skim-milk flocculation) and detection methods (immunofluorescence assay, cell culture, polymerase chain reaction (PCR), integrated cell culture PCR (ICC-PCR), and quantitative PCR) have been developed and demonstrated for HPyV, which has enabled the identification and quantification of HPyV in various environmental samples, such as sewage, surface water, seawater, drinking water, and shellfish. In this paper, we summarize these recent advancements in detection methods and the accumulation of environmental surveillance and laboratory-scale experiment data, and discuss the potential advantages as well as limitations of HPyV as a human-specific viral marker in aquatic environments.

Enteric and indicator virus removal by surface flow wetlands.

We investigated the occurrence and attenuation of several human enteric viruses (i.e., norovirus, adenovirus, Aichi virus 1, polyomaviruses, and enterovirus) as well as a plant virus, pepper mild mottle virus (PMMoV), at two surface flow wetlands in Arizona. The retention time in one of the wetlands was seven days, whereas in the other wetland it could not be defined. Water samples were collected at the inlet and outlet from the wetlands over nine months, and concentration of viral genomes was determined by quantitative polymerase chain reaction (qPCR). Of the human enteric viruses tested, adenovirus and Aichi virus 1 were found in the greatest prevalence in treated wastewater (i.e., inlet of the wetlands). Reduction efficiencies of enteric viruses by the wetlands ranged from 1 to 3 log10. Polyomaviruses were generally removed to below detection limit, indicating at least 2 to 4 log10 removal. PMMoV was detected in a greater concentration in the inlet of both wetlands for all the viruses tested (10(4) to 10(7) genome copies/L), but exhibited little or no removal (1 log10 or less). To determine the factors associated with virus genome attenuation (as determined by qPCR), the persistence of PMMoV and poliovirus type 1 (an enterovirus) was studied in autoclaved and natural wetland water, and deionized water incubated under three different temperatures for 21 days. A combination of elevated water temperature and biological activities reduced poliovirus by 1 to 4 log10, while PMMoV was not significantly reduced during this time period. Overall, PMMoV showed much greater persistence than human viruses in the wetland treatment.

Quantification and genetic analysis of salivirus/klassevirus in wastewater in Arizona, USA.

Salivirus/klassevirus sequences were identified in 7 (15%) wastewater samples collected in Arizona monthly for a year, with the highest concentration of 2.28 × 10(5) and 2.46 × 10(4) copies/L in influent and effluent, respectively. This is the first report of quantification and genetic analysis of salivirus/klassevirus in water samples in the United States.