Decay and solid-liquid partitioning of mpox and vaccinia virus DNA in primary influent and settled solids to guide wastewater-based epidemiology practices.

Wastewater-based epidemiology (WBE) has proven to be a powerful tool for tracking the spread of viral pathogens, such as SARS-CoV-2; however, as WBE has expanded to include new pathogens, like mpox virus (MPXV), more data is needed to guide practitioners on how to design WBE campaigns. Here, we investigated the decay rates of heat-inactivated MPXV (HI-MPXV) and attenuated vaccinia virus (VV) viral signal in primary influent and settled solids collected from a local POTW at 4 °C, 22 °C, or 35 °C using digital PCR. Subsequently, we studied the solid-liquid partitioning of the viruses in primary influent. Over 30 days, decay rates did not significantly differ between viruses (p = 0.5258). However, decay was significantly higher in primary influent (0.109-0.144/day) than in settled solids (0.019-0.040/day) at both 22 °C (p = 0.0030) and 35 °C (p = 0.0166). Furthermore, as part of the partitioning experiment, we found that HI-MPXV and VV adsorb to the solids fraction of primary influent at higher intensities than previously studied enveloped viruses (KF = 1000-31,800 mL/g, n = 1.01-1.41). Likewise, it was determined in the partitioning experiment that a concentration of greater than 103 gc/mL in raw influent was needed for the viable quantification of MPXV and VV DNA in the clarified liquid fraction of raw primarily influent. Our study provides essential insights into informative sample collection and storage conditions for the analysis of wastewater and for transport modeling studies. Due to the slow decay observed in settled solids at all tested temperatures in the persistence experiment, this matrix may be most suitable for retrospective analyses of MPXV infections within a community.