Suspended Solids and Optimal RNase Inhibitors Impact the Partitioning and Decay of SARS-CoV-2 in Wastewater

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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus causing COVID-19, can be monitored in wastewater due to its presence in human fecal matter. While wastewater surveillance programs for COVID-19 have already been implemented in many countries, fundamental questions remain regarding the distribution and decay of both SARS-CoV-2 and the common fecal indicator pepper mild mottle virus (PMMoV). In this study, for wastewater samples at 4 °C, the first-order decay rate constant ( k ) for a spiked coronavirus (HCoV 229E) was greater in mixtures with low TSS concentrations (0.373 ± 0.021 day –1 ) than in those with high concentrations (0.204 ± 0.014 day –1 ), which was consistent with measurements of the extended activity of RNases. Increasing the concentration of nontargeted and targeted RNase inhibitors revealed that the loss of the viral signal from the extraction is mainly due to the activity of RNA-degrading enzymes. A reanalysis of wastewater samples from Quebec City, Canada, with a 10× concentration of β-mercaptoethanol, a nontargeted RNase inhibitor, achieved an increase in SARS-CoV-2 and PMMoV concentrations. This investigation revealed further optimization avenues for improving the detection limit of SARS-CoV-2 in wastewater and enhancing the efficiency of wastewater surveillance programs, particularly in times of low viral prevalence.