West Nile Virus (

Background: Methods: A targeted droplet digital reverse transcription-polymerase chain reaction (RT-PCR) approach (ddRT-PCR) was used to measure WNV concentrations in wastewater retrospectively from five locations and in over 600 samples. Three of these locations were in communities with multiple confirmed WNV infections, while two were not. Samples were collected during periods corresponding to typical WNV seasonality (spring to fall). SARS-CoV-2 RNA was measured simultaneously to assess nucleic acid degradation during sample storage. Publicly available confirmed WNV case data were compiled from the California and Nebraska departments of public health and their weekly arboviral reports. Results: WNV RNA was detected in wastewater samples during periods of known viral circulation within a community. The adopted ddRT-PCR assay is highly specific and sensitive, and detections in wastewater solids correspond to the occurrence of cases in the season and location of sampling. WNV was detected in nine samples in three locations with known WNV clinical cases-wastewater positivity rates in these locations ranged from 3.3% to 13%. The results suggest that wastewater monitoring could serve as an effective complement to traditional surveillance methods, particularly for sentinel surveillance in locations which do not have extensive mosquito and clinical testing systems.