*4.4. Real-Time RT-PCR*

Brain of horses and birds, EDTA blood, and CSF suspected to be infected with WNV were stored at −80 ◦C until analysis. Brains were grinded in Dulbecco modified Eagle's minimal essential medium (DMEM) with ceramic beads (MP Biomedicals, Illkirch, France) and FastPrep ribolyzer in BSL3 facilities. A total of 560 μL of Lysis bu ffer from the QIAamp Viral RNA kit (Qiagen, Hilden, Germany) were added to 140 μL of grinded material before RNA extraction with the automate QIAcube. Human samples (EDTA blood and CSF) were processed the same way. Every RNA extracts were subjected to real time (rt) RT-PCR following the protocol described earlier by Linke et al. [68].

### *4.5. Virus Isolation*

One milliliter of brain homogenates of WNV rtRT-PCR positive wild birds and horses was prepared in DMEM culture medium and inoculated on T25 flask that had been seeded with Vero NK cells (ATCC: CCL81™), 24 h earlier and washed with DMEM before inoculation. After 1h 30 of incubation at 37 ◦C with 5% CO2, cells were washed twice with phosphate buffered saline (PBS), and complete medium (DMEM+ 1% penicillin- streptomycin+ 1% sodium pyruvate + 5% fetal calf serum) was added. The cells were observed each day from 3 days to 7 days post infection (pi). As soon as cytopathic effects (CPE) were detected, the supernatant was collected, stored at −80 ◦C, and RNA extracts subjected to rtRT-PCR to confirm WNV detection. Primary isolation was followed by a passage on Aedes albopictus (C6/36) (ATCC® CRL1660™) cell line. A total of 200μL−1 mL of Vero cell supernatants was added to T25 flask that had been seeded with C6/36 24 h earlier and washed with Leibowitz L15 medium before inoculation. After 1h 30 of incubation at 28 ◦C without CO2, 6 mL of Leibowitz L15 media + 1% penicillin- streptomycin+ 1% sodium pyruvate + 1% L Glutamin+ 10% fetal calf serum were added. CPEs were not systematically observed in C6/36 cells and supernatants were collected on day 7 post-infection at the latest and tested as described above. This protocol is adapted from the OIE Manual of Diagnostic Tests and vaccines for Terrestrial Animals [69].

### *4.6. Nucleotide Sequencing and Sequence Analysis*

Sequencing libraries were prepared from genomic RNAs extracted from virus isolate (2015) or from organ homogenates (2018) and whole-genome sequencing data were obtained as previously described (Ion Torrent sequencing and assembly with CLC Genomics Workbench for Genbank accession number MT863559, 2015 [70] or with bwa for Genbank accession numbers MT863560-1, 2018 [71]). Multiple alignment of the nucleotide sequences was performed using the ClustalW algorithm and phylogenetic analysis was performed using the Neighbor–Joining and Maximum Likelihood methods in MEGA7 [36].
