*4.3. Sample Collection*

Detailed necropsies were performed on the euthanized individuals. Samples of the brain, heart, lung, liver, spleen, kidney, thymus, bursa of Fabricius, and feather pulp were collected into sterile polypropylene tubes filled with 1 mL of Hanks' balanced solution (10% glycerol, 200 U/mL penicillin, 200 μg/mL streptomycin, 100 U/mL polymixin B sulphate, 250 μg/mL gentamicin, and 50 U/mL nystatin) and stored at −70 ºC until analysis by real-time reverse transcription polymerase chain reaction (RRT-PCR). In addition, samples of the brain, oral mucosa, thymus, heart, trachea, lung, liver, spleen, kidney, small and large intestine, pancreas, cecal tonsils, bursa of Fabricius, pectoral muscle, and skin with feather follicles were fixed in 10% neutral buffered formalin.

### *4.4. Virus Genome Detection*

RNA was extracted from tissue samples after homogenization and tested by RRT-PCR for the presence of the WNV genome as described in Sotelo et al. [17].
