Usutu virus (USUV) forward primer complementing the Jost et al. primer set

We designed an additional forward primer, USUV_MM_for, to detect Usutu virus (USUV) in diagnostic samples that is compatible with the reverse primer as well as the probe of the primer pair published by Jöst et al. (2011). The combined primer pair produces a PCR product of 265 bp, making it more suitable for Sanger sequencing downstream. The primer pair was designed by aligning different USUV strains, including Africa 1 (KC754958), Africa 2 (AY453412), Africa 3.1 (MN122238), Africa 3.2 (MN122249), Europe 3 (MN122196) and WNV Lin1 (MW627239), selecting a region of the genome that is conserved for USUV but divergent for WNV. RT-qPCR analysis showed that the new forward primer (combined with the reverse Jost primer) amplified USUV but not WNV (lin 1 and lin 2) or RVFV (PCR report and gel image attached).