Evaluation of pepper mild mottle virus as an indicator of human faecal pollution in shellfish and growing waters

Bivalve molluscan shellfish grown in areas impacted by human faecal pollution are at risk of being contaminated with multiple enteric viruses. To minimise the public health risks associated with shellfish consumption, determining the presence of faecal contamination in shellfish and their growing waters is crucial. In this study, we evaluated the use of pepper mild mottle virus (PMMoV) as an indicator of human faecal contamination in oysters, mussels, cockles and shellfish growing waters in New Zealand. Using reverse transcription quantitative polymerase chain reaction (RT-qPCR) the presence, and where applicable, the concentration of PMMoV was determined in faeces from 11 different animal species, influent (untreated) wastewater, shellfish and shellfish growing waters. Non-human faecal samples (from seagull, Canada goose, black swan and dog) were RT-qPCR positive for PMMoV. The faecal source specificity of PMMoV was 0.83 (maximum value of 1) when 'detected but not quantifiable' (DNQ) values were used. However, when 'lower limit of quantification' (LLOQ) values were used, the specificity increased to 0.92. The PMMoV concentration in influent wastewater (n = 10) ranged from 6.3 to 7.7 log₁₀ genome copies (GC)/L with a mean (±standard deviation) of 7.1 ± 0.5 log₁₀ GC/L. The overall occurrence of PMMoV in shellfish and shellfish growing waters from four different areas was 46/51 (90%) and 29/52 (56%), respectively. Of the cockles collected from an area known to be impacted by effluent wastewater, 14/14 (100%) contained PMMoV concentrations above the LLOQ. In contrast, only 13/37 (35%) shellfish and 6/52 (11.5%) growing water samples collected from three areas with low anthropogenic impact contained PMMoV concentrations above the LLOQ. The high concentration of PMMoV in influent wastewater indicates that PMMoV may be a promising indicator of human faecal contamination. The presence of PMMoV in shellfish and growing waters with a low anthropogenic impact may be of avian origin, and this needs to be considered if using PMMoV for monitoring shellfish and shellfish growing water quality in New Zealand.