Systematic Comparison of Indicator and Pathogenic Viruses Using High-Throughput qPCR Identifies Pepper Mild Mottle Virus as a Robust Indicator of Virus Removal in Wastewater Treatment

The selection of appropriate viral indicators for evaluating wastewater treatment performance remains challenging because candidate markers have rarely been compared systematically within a unified analytical framework. Here, we collected influent and effluent samples monthly for one year from two wastewater treatment plants in Japan and conducted, to our knowledge, the first comprehensive comparison of 19 viral targets and one protozoan target using high-throughput quantitative PCR. Pepper mild mottle virus (PMMoV) was consistently detected at high concentrations, showed limited seasonal variability, and exhibited an approximately 1.0 log10 reduction, comparable to those observed for pathogenic viruses. In contrast, Carjivirus, formerly known as crAssphage, was present at the highest concentrations but showed significantly greater reduction than pathogenic viruses. Tomato brown rugose fruit virus (ToBRFV), despite its high abundance and emerging recognition as a potential marker, exhibited pronounced seasonal fluctuations. Other Tobamovirus species, such as cucumber green mottle mosaic virus and tobacco mild green mosaic virus, exhibited similar removal but lower prevalence compared with PMMoV. Overall, PMMoV demonstrated the most balanced performance in terms of abundance, stability, and removal behavior, supporting its use as a robust indicator for monitoring virus removal in wastewater treatment.