Viral tissue burden and viremia quantification
For in vivo virus replication experiments, mice were infected with WNV and euthanized at specific days post-infection, as indicated. For tissue collection, mice were deeply anesthetized with ketamine/xylazine/acepromazine, blood collected in serum separator tubes, then transcardially perfused with sterile Dulbecco’s phosphate-buffered saline (dPBS; Gibco). The spleen and kidneys were collected, then the brain and spinal cord removed and microdissected. All organs were snap frozen, weighed, and then homogenized in dPBS. Virus was titered by standard plaque assay with BHK21 cells, as described previously [46]. Serum viremia was measured using TaqMan quantitative RT-PCR (qRT-PCR) primers and probe listed in Table 1, as described previously [47].
Table 1 Primer and probe sequences used for qRT-PCR