Objective To establish a stably passaged tree shrew aortic endothelial cell line (TSAEC), and to explore its application in respiratory syncytial virus (RSV) infection research.

Methods Primary aortic endothelial cells derived from tree shrews were isolated and cultured using the tissue explant adherence method, and an immortalized TSAEC was established through lentivirus-mediated SV40T gene transduction. Immunofluorescence staining was performed to detect the expression of endothelial cell markers, including von Willebrand factor (vWF) and platelet endothelial cell adhesion molecule-1 (CD31), to characterize the cellular phenotype. Cell proliferative activity and passage stability were assessed using the Cell Counting Kit-8 (CCK-8) assay. Furthermore, tree shrew lung fibroblasts (TSLFC), tree shrew vascular smooth muscle cells (TSVSMC), human laryngeal epidermoid carcinoma cells (Hep-2), and African green monkey kidney cells (Vero) were used as control cells to compare RSV replication capacity among different cell types following infection. Viral susceptibility of TSAEC was evaluated by measuring viral load, viral titer, and observing cytopathic effects.

Results The isolated TSAECs showed a typical flat spindle shape and highly expressed vascular endothelial marker proteins vWF and CD31. Cell proliferation activity was detected by CCK-8 assay. The results showed that there was no significant difference in proliferation capacity between untransfected primary cells and P10 generation cells. All generations of cells entered the logarithmic growth phase in the middle of culture and still maintained stable cell morphology and high cell viability till P60 generation TSAECs. Compared with TSLFC, TSVSMC, Hep-2 and Vero cells, RSV had stronger replication ability in TSAECs, and typical syncytial-like lesions were observed after infection.

Conclusion This study successfully established the immortalized TSAEC, which was highly susceptible to RSV and could be used for the research on RSV infection mechanism and antiviral medicine.

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