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

ABSTRACT We investigated the potential use and quantitation 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 and RNA enteric viruses, 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 quantitated in the extracted samples via quantitative PCR using TaqMan assays. The average gene copies assessed in the raw sewage were not significantly different (p-values ranged between 0.0547 and 0.7986) than the average gene copies assessed in the effluents for Adenovirus and crAssphage (DNA viruses), Pepper Mild Mottle Virus (RNA virus), and uidA in terms of both volume and biomass. A significant reduction of these enteric viruses was observed consistently in activated sludge samples compared with those for raw sewage. Corresponding reductions in gene copies per volume and gene copies per biomass were also seen for uidA but were not statistically significant (p-value = 0.8769 and p-value = 0.6353, respectively). The higher gene copy numbers of enteric viruses and E. coli observed in the effluents may be associated with the 12-hour hydraulic retention time in the facility. Enteric viruses found in gene copy numbers were at least one order of magnitude higher than the E. coli marker uidA. This indicate that enteric viruses may survive the wastewater treatment process and viral-like particles are being released into the aquatic environment. Our results suggest that Adenovirus, crAssphage, and Pepper mild mottle virus can be used as complementary viral indicators of human fecal pollution.