Quantification of human enteric viruses as alternative indicators of fecal pollution to evaluate wastewater treatment processes

Abstract

We investigated the potential use and quantification of human enteric viruses in municipal wastewater samples of Winnipeg (Manitoba, Canada) as alternative indicators of contamination and evaluated the processing stages of the wastewater treatment plant. During the fall 2019 and winter 2020 seasons, samples of raw sewage, activated sludge, effluents, and biosolids (sludge cake) were collected from the North End Sewage Treatment Plant (NESTP), which is the largest wastewater treatment plant in the City of Winnipeg. DNA (Adenovirus and crAssphage) and RNA enteric viruses (Pepper mild mottle virus, Norovirus genogroups GI and GII, Rotavirus Astrovirus, and Sapovirus) as well as the uidA gene found in Escherichia coli were targeted in the samples collected from the NESTP. Total nucleic acids from each wastewater treatment sample were extracted using a commercial spin-column kit. Enteric viruses were quantified in the extracted samples via quantitative PCR using TaqMan assays. Overall, the average gene copies assessed in the raw sewage were not significantly different (p-values ranged between 0.1023 and 0.9921) than the average gene copies assessed in the effluents for DNA and RNA viruses and uidA in terms of both volume and biomass. A significant reduction (p-value ≤ 0.0438) of Adenovirus and Noroviruses genogroups GI and GII was observed in activated sludge samples compared with those for raw sewage per volume. Higher GCNs of enteric viruses were observed in dewatered sludge samples compared to liquid samples in terms of volume (g of sample) and biomass (ng of nucleic acids). Enteric viruses found in gene copy numbers were at least one order of magnitude higher than the E. coli marker uidA, indicating that enteric viruses may survive the wastewater treatment process and viral-like particles are being released into the aquatic environment. Viruses such as Noroviruses genogroups GI and GII, and Rotavirus were detected during colder months. Our results suggest that Adenovirus, crAssphage, and Pepper mild mottle virus can be used confidently as complementary viral indicators of human fecal pollution.

Development and evaluation of TaqMan-based, one-step, real-time RT-PCR assays for pepper mild mottle virus detection for near source tracking and wastewater-based epidemiology validation

“…Continuous sampling technologies are at the cutting edge of wastewater epidemiology and our validation strategy can be applied to these new technologies. Studies [17][18][19] published to date using Taq-Man assays to identify PMMoV have based their TaqMan primer/probe sequences on two studies [13,14]. Haramoto et al modified their forward primer from the Zhang et al study to correct for a nucleotide mismatch at the 3' end to attain full primer-target sequence matching, highlighting that TaqMan assays can be improved upon with subsequent redesign.…”

Seasonal Patterns of Viromes in Urban Aquatic Environments of Manitoba

“…Assembly Phage (crAssphage), Pepper mild mottle virus (PMMoV) and Rotavirus (RoV), as previous literature and results in the Uyaguari laboratory has evidenced these enteric viruses to be relatively more stable enteric viral markers of fecal contamination (Garcia et al, 2022). Using the results generated from these approaches, seasonal patterns of viral communities were then determined using exploratory factor analysis, distance metrics on the beta diversity index, and generalized linear model (GLM) on the alpha diversity indices.…”

“…Even though treated water samples tested negative for the presence of E. coli and total coliforms, these outbreaks raised questions about the sole usefulness of the current microbial standards of aquatic health for routine wastewater quality testing. Aside from Norovirus outbreaks, there are many other reports on Rotavirus, crAssphage, and Adenovirus prevalence in freshwaters (Hellmer et al, 2014; Garcia et al, 2022; Mafumo et al, 2023).…”

Virus inactivation by sequential ultraviolet-chlorine disinfection: Synergistic effect and mechanism

“…SARS-CoV-2 has been detected in both wastewater ( Elsamadony et al, 2021 ; La Rosa et al, 2021 ; Mota et al, 2021 ) and surface water ( Guerrero-Latorre et al, 2020 ). Additionally, various enteric viruses, such as adenovirus (AdV), enterovirus (EV), norovirus (NoV), hepatitis A virus (HAV) and rotavirus (RV), have been found in diverse aquatic environments ( Borchardt et al, 2012 ; Wong et al, 2012 ; Ye et al, 2012 ; Mackowiak et al, 2018 ; Sedji et al, 2018 ; Chacon et al, 2020 ; Sorensen et al, 2021 ; Garcia et al, 2022 ). For example, AdV, EV, and NoV GⅠ were shown to be present in municipal wastewater at concentrations up to 7.91–9.00 log 10 copies L −1 ( da Silva et al, 2007 ; Hewitt et al, 2011 ; O'Brien et al, 2017 ).…”