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Primers and the probe of assay 1 target the 5\u2032-untranslated region (UTR), whereas the amplicon of assay 2 is located in the nonstructural region NS2A, which enables an unambiguous and independent WNV diagnosis based on 2 different amplicons. Both assays allow the detection of as few as 2\u20134 genome copies of WNV strains NY99, Uganda B956, Kunjin, and Sarafend (all cultured on Vero cells). 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