A New Fluorogenic Real-Time RT-PCR Assay for Detection of Lineage 1 and Lineage 2 West Nile Viruses

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Abstract
West Nile virus represents an emerging threat for animal and human health worldwide. This virus exhibits a marked genetic variation, with at least 2 distinct evolutionary lineages. Lineage 1 has been recognized in Africa, Asia, Europe, Oceania, and more recently in the Americas, whereas lineage 2 is restricted to Africa. Perhaps for this reason, the available real-time RT-PCR methods for detecting West Nile virus genome have mainly focused on lineage 1. However, both viruses may potentially be spread beyond their endemic areas by migratory birds. This report describes a new real-time reverse transcription-PCR (RT-PCR) method based on a 59-Taq nuclease-39 minor groove binder DNA probe (TaqMan MGBE) that allows the detection of a wide range of West Nile virus isolates, including both lineages 1 and 2. This method was able to detect West Nile viruses from different origins (North and Central Africa, Middle East, Europe, and North America), whereas other flaviviruses (Usutu, Dengue, Yellow fever) analyzed in parallel remained negative. The sensitivity achieved by this assay was 1022-1023 pfu/tube. This method, which can be performed in 96-well format, could be suitable for the large-scale surveillance of West Nile virus in areas where both lineages can potentially spread.