Establishment of an immortalized Muscovy duck embryo fibroblast cell line and exploration of virus tropism

Muscovy duck embryo fibroblasts (MDEF) are a useful cellular model for studying the molecular mechanisms associated with waterfowl viruses infection. However, the short lifespan and poor passaging capacity of MDEF limit their application. This study aimed to establish an immortalized MDEF cell line (iMDEF) by transfection the simian virus 40 large T antigen (SV40 LT) gene with immortalization function into primary MDEFs. After transfection with SV40 LT lentiviral vector and screening with puromycin, iMDEF cell line was developed with stable and high expression of SV40 LT gene. This iMDEF cell line maintained stable fibroblast morphology and proliferated continuously for over 50 passages. Compared with the primary MDEF, the proliferation of iMDEF cells was significantly enhanced, the growth kinetics was stable, and the apoptosis rate was low. Safety assessments confirmed that there were no endogenous waterfowl viruses in iMDEFs, and iMDEFs had no tumorigenicity in nude mice. Importantly, various waterfowl viruses could replicate well in iMDEFs, and here is no difference in the replication ability as compared to primary MDEFs. In conclusion, we successfully developed a stable and non-tumorigenic iMDEF cell line, overcoming the limitations of primary MDEFs. This cell model provides a consistent and cost-effective platform for studying waterfowl virus-host interactions, vaccine development, and viral pathogenic mechanisms.