Diagnostic testing

Whole (EDTA) blood was collected for the detection of virus by an in-house developed real-time reverse transcriptase PCR (rRT-PCR) detecting all 24 traditional serotypes of BTV [21]. From the EDTA blood, isolated dsRNA samples were tested using primers (Eurogentec Nederland b.v., Maastricht, Netherlands), the forward primer is 5’-AGTGTCGCTGCCATGCTATC-3’ and the reverse primer is 5’-GCGTACGATGCGAATGCA-3’, and a Taqman probe 5’-6FAM-CGAACCTTTGGATCAGCCCGGA-XTMR-PH (Tib MolBiol, Berlin, Germany) targeting segment 10 of the BTV genome. RRT-PCR was performed by use of the LightCycler RNA Master Hybridization Probes kit (Kit, Roche Diagnostics Nederland b.v., Almere, Netherlands) with a LightCycler 2.0 (Roche Diagnostics Nederland BV, Almere, Netherlands). Three positive controls were included containing different virus dilutions of BTV1 grown on BHK21 cells in DMEM with 5% fetal bovine serum and diluted in the same growth medium. A run was successful when all negative controls were negative, and positive controls were positive. Results of test samples were considered positive if the software generated a crossing point (cp) value and a sigmoid curve with a signal (OD530/OD640) at least partly above the cut-off value. Results were considered doubtful in case of a sigmoid-shaped curve completely below the cut-off value, or in case of all other not interpretable curves.

Furthermore, the sera were tested for BTV specific antibodies in a commercial competitive ELISA (ID Screen® bluetongue Competition ELISA kit, ID Vet, Montpellier, France) according to manufacturer’s instructions. Results were measured as binary variable: positive or negative.

The test results, i.e. the data on which our analyses were based, are available as S1 File.