Detection of West Nile virus in large pools of mosquitoes

We conducted a laboratory evaluation of the ability of commercial antigen-capture assays, the Rapid Analyte Measurement Platform (RAMP (R)) and the VecTest (R) wicking assay, as well as Real Time reverse transcriptase-polymerase chain reaction (RT-PCR, Taqman) and Vero cell plaque assay to detect West Nile virus (WN-V) in large mosquito pools. Real-Time PCR (Taqman) was the most sensitive, detecting WN-V ribonucleic acid (RNA) in 100% of samples containing a single infected mosquito in pool sizes of up to 500 mosquitoes. Mosquito body tissues minimally impacted the ability of Real Time RT-PCR to detect WNV in a pool size of 500, reducing sensitivity by 0.6 log(10) plaque-forming units (PFU)/ml. Vero cell plaque assay detected live virus from a single infected mosquito in 100% of pools containing up to 200 mosquitoes, but was unreliable at larger pool sizes. VecTest detected 100% of positive pools containing 50 mosquitoes with 5.8 log(10) PFU/ml virus, 100 mosquitoes with 5.9 log(10) PFU/ml, and 200 mosquitoes with 5.2 log(10) PFU/ml, The RAMP assay detected 100% of positive pools containing 50 mosquitoes with 3.3 log(10) PFU/ml virus, 100 mosquitoes with 3.7 log(10) PFU/ml, and 200 mosquitoes with 4.0 log(10) PFU/ml. Results indicate that WN-V can be reliably detected by all 4 assays in pools of mosquitoes exceeding 50 specimens, though there is some loss of sensitivity with very large pool sizes.