USUV Detection in Blackbird Samples

For USUV, molecular detection was performed as described previously by Oude Munnink et al. 2020 [33 (link)]. Brain tissue samples (N = 200) collected from blackbirds during post-mortem investigation were homogenized in 300uL tissue lysis buffer (MagNA Pure DNA Tissue Lysis Buffer) using the Fastprep bead beater (4.0 m/s for 10 s), RNA extraction and USUV RT-PCRs were performed with an additional dilution step, where 60uL homogenized sample was added to 540uL VTM (or DMEM) and 600uL External lysis buffer. Phocine distemper virus (PDV) was used as an internal control [34 (link),35 (link)]. Throat and cloaca swabs of live blackbirds were stored in Virus Transport Medium (VTM). 600 uL of MagNA Pure 96 External Lysis Buffer (Manufactured by Roche: https://lifescience.roche.com/en_nl/products/magna-pure-96-external-lysis-buffer.html) was added to 600 uL of sample and RNA extraction was performed on a MagNA Pure 96 Instrument (MagNA Pure 96 Instrument (roche.com)). USUV RT-PCRs [34 (link),35 (link)] were performed, using Phocine distemper virus (PDV) as an internal control. For USUV investigation, mosquito pools were tested according to the protocol reported by Blom et al., 2023 [36 (link)].