Propagation of Recombinant Newcastle Disease Viruses

Chicken embryonic fibroblasts (DF-1) were purchased from the Cell Resource Center of Shanghai Institutes for Biological Sciences of the Chinese Academy of Sciences. BSR-T7/5 cells stably expressing the T7 phage RNA polymerase were a kind gift from Prof. Zhigao Bu (Harbin Veterinary Research Institute, China). DF-1 and were BSR-T7/5 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics and were cultured at 37 °C under 5% CO₂. The parental NDV (rSS1GFP) and the mutant NDV harboring NLS mutation in the M protein (rSS1GFP-M/NLSm) were generated in our previous study [22 (link)]. The two viruses were plaque purified three times in DF-1 cells and propagated once in specific-pathogen-free (SPF) embryonated chicken eggs. Primary antibody mouse anti-Tubulin monoclonal antibody (sc-53646), mouse anti-GAPDH monoclonal antibody (sc-66163), mouse anti-Lamin B1 monoclonal antibody (sc-56143), and mouse anti-GFP monoclonal antibody (sc-9996) were purchased from Santa Cruz Biotechnology (USA). Mouse anti-PROX1 monoclonal antibody (ab33219), and rabbit anti-AHR polyclonal antibody (ab84833) were purchased from Abcam (UK).