Comparing Recovery Methods for Wastewater Surveillance of Arthropod-Borne and Enveloped Viruses

The potential public health utility of arthropod-borne virus (arbovirus) wastewater surveillance (WWS) warrants the effort of establishing an effective sample preparation workflow. Here, we investigated different sample clarification, concentration, and extraction methods to determine the best workflow for recovery of spiked arboviral and murine hepatitis virus RNA signals in wastewater. We determined that the sample clarification method with the least loss is filtration through a 0.22 μm filter (9.06 ± 3.68%) when compared with centrifugation. Out of four different concentration methods (centrifugal ultrafiltration, polyethylene glycol precipitation, charged membrane capture and elution, and hollow-fiber ultrafiltration), centrifugal ultrafiltration, specifically using Macrosep Advance column (30 kDa), has the highest mean recovery of 84.76 ± 17.45%. With this method, we can reliably recover seeded arboviruses at concentrations corresponding to 10 infections in 400 to 180,000 people, depending on the shedding rate. As arboviruses are expected to be on the rise due to the expansion of their vectors’ geographic distribution, this research will lay the foundation for managing and reducing the impact of arboviral outbreaks through WWS-informed public health measures.