Development of a Rapid, Cost-Effective, and Automatable Virus Concentration from Wastewater Using a Coagulant with Magnetic Materials

Wastewater-based epidemiology (WBE) is recognized as an effective approach for monitoring infectious diseases and informing public health decisions. However, conventional virus concentration methods, such as polyethylene glycol (PEG) precipitation or membrane filtration, can be labor-intensive and difficult to automate for large-scale studies. In this work, we evaluated rapid and centrifugation-free virus concentration strategies to enable high-throughput WBE. First, we compared flocculation using polyaluminum chloride (PAC) and poly-γ-glutamic acid (polyglu) with established methods (PEG precipitation and low-speed centrifugation). Polyglu and PAC both showed high transfer rates (over 80%) of enveloped (SARS-CoV-2, MHV) and nonenveloped (PMMoV, MNV) viruses into the floc. Despite slight reductions in overall recovery from the sediment fraction, PAC enabled robust floc formation and faster processing than PEG. Next, we optimized lysis buffer conditions and found that neutral buffers (e.g., PBS, glycine pH 7.5) maximized RNA recovery from PAC-containing sediments. Finally, incorporating ferrous ferric oxide into PAC-based flocs allowed efficient magnetic separation, eliminating the need for centrifugation and achieving comparable viral recovery rates. This study demonstrates the feasibility of a magnetically assisted flocculation approach to concentrate diverse viruses from wastewater, offering a promising step toward a fully automated and high-throughput WBE platform.