H9N2 Virus Infection and PD-L1 Induction

The H9N2 virus (Ck/HB/4/08) was inoculated into 9-day-old SPF chicken embryos, and virus titers were determined by measuring PFUs. Madin-Darby canine kidney (MDCK, CCL-34, ATCC) cells used for PFUs were cultured in DMEM media supplemented with 5% FBS. AIV-specific sialic acid α-2,3-galactose receptor (SA2-3Gal) expression was confirmed by biotinylated Maackia amurensis lectin II (VECTOR, CA, USA) staining and then followed by staining with FITC-conjugated avidin D (green) and DAPI (blue) for nuclei. To assess H9N2 virus infection, RPMECs were washed with PBS, inoculated with virus at different multiplicities of infection (MOIs) and incubated for 1 h. Then, the cells were washed with PBS and incubated with DMEM, 0.2% bovine serum albumin (Gibco, Carlsbad, CA, USA) and 0.2 μg/mL TPCK-treated trypsin [20 (link)]. Viral titers in the supernatants were measured using PFUs. To investigate the PD-L1 level induced by inactivated H9N2 virus, viral particles were inactivated using 0.094% β-propionolactone (BPL; SERVA Electrophoresis, Heidelberg, Germany) according to a previously described protocol [21 (link)].