Suspended Solids\nand Optimal RNase Inhibitors Impact\nthe Partitioning and Decay of SARS-CoV‑2 in Wastewater

Severe acute respiratory syndrome\ncoronavirus 2 (SARS-CoV-2),\nthe\nvirus causing COVID-19, can be monitored in wastewater due to its\npresence in human fecal matter. While wastewater surveillance programs\nfor COVID-19 have already been implemented in many countries, fundamental\nquestions remain regarding the distribution and decay of both SARS-CoV-2\nand the common fecal indicator pepper mild mottle virus (PMMoV). In\nthis study, for wastewater samples at 4 °C, the first-order decay\nrate constant (<i>k</i>) for a spiked coronavirus (HCoV\n229E) was greater in mixtures with low TSS concentrations (0.373 ±\n0.021 day<sup>–1</sup>) than in those with high concentrations\n(0.204 ± 0.014 day<sup>–1</sup>), which was consistent\nwith measurements of the extended activity of RNases. Increasing the\nconcentration of nontargeted and targeted RNase inhibitors revealed\nthat the loss of the viral signal from the extraction is mainly due\nto the activity of RNA-degrading enzymes. A reanalysis of wastewater\nsamples from Quebec City, Canada, with a 10× concentration of\nβ-mercaptoethanol, a nontargeted RNase inhibitor, achieved an\nincrease in SARS-CoV-2 and PMMoV concentrations. This investigation\nrevealed further optimization avenues for improving the detection\nlimit of SARS-CoV-2 in wastewater and enhancing the efficiency of\nwastewater surveillance programs, particularly in times of low viral\nprevalence.