Genetic Evaluation of Pepper Mild Mottle Virus as an Indicator in Water Quality Monitoring and Human Fecal Contamination in Swedish Waters

Pepper mild mottle virus (PMMoV) has emerged as a promising viral indicator for human fecal contamination and evaluating viral removal efficiency in water treatment systems. However, few studies have examined this potential from a genetic perspective over extended timescales. This study investigated PMMoV presence and genetic diversity across multiple water environments in Sweden, including wastewater, lake water, and raw water used for drinking water production, collected between 2016 and 2024. PMMoV was consistently detected in both wastewater and lake water, showing relatively stable concentrations in wastewater between years, and with levels typically 4-7 log₁₀ higher than in lake water. Comparative genotyping of the coat protein (CP) and replication-associated (RP) regions for wastewater samples showed no significant differences. We successfully sequenced 67 isolates from wastewater, 16 from lake water, and 2 from source water at the CP region. Sequence analysis revealed extremely high nucleotide identities (> 96%) with minimal divergence between samples collected six years apart or from different water types. Phylogenetic comparisons with global reference sequences confirmed high genetic conservation worldwide. These findings demonstrate the remarkable genetic stability of PMMoV over time and geography, supporting its reliability as a viral process indicator for water treatment systems. However, the potential for PMMoV to originate from non-human sources suggests it should not be used as a standalone marker for human-specific fecal contamination. Overall, this study highlights the global genetic conservation of PMMoV and its utility for long-term environmental surveillance and water quality assessment.