2.3. Nucleic Acid Amplification

Extracted nucleic acid solutions were tested for the presence of WNV-RNA using two published RT-qPCRs [50], one targeting the 5’-untranslated region (INEID) and one the nonstructural NS2A protein gene region (WNF-FLI). Samples were also tested for Usutu virus (USUV) using a USUV-specific RT-qPCR, targeting a nonstructural protein 1 gene region [51]. For all RT-qPCRs, either the AgPath-ID One-Step RT-PCR Kit (ThermoFisher Scientic) or the QuantiTect Probe RT-PCR Kit (Qiagen) was used. Realtime PCRs were run on a CFX96 RealTime System (Bio-Rad, Munich, Germany).

Nucleic acids extracted from dilution series of WNV strains 203204 (GenBank accession number: {"type":"entrez-nucleotide","attrs":{"text":"JF719066","term_id":"341869843","term_text":"JF719066"}}JF719066), 204913 ({"type":"entrez-nucleotide","attrs":{"text":"KF114267","term_id":"588996858","term_text":"KF114267"}}KF114267), NY99 ({"type":"entrez-nucleotide","attrs":{"text":"AF196835","term_id":"11597239","term_text":"AF196835"}}AF196835) and Austria 361/10 ({"type":"entrez-nucleotide","attrs":{"text":"HM015884","term_id":"300827484","term_text":"HM015884"}}HM015884), and from USUV strain Europa 3 ({"type":"entrez-nucleotide","attrs":{"text":"HE599647","term_id":"347977231","term_text":"HE599647"}}HE599647), were used as positive controls.