Abstract
Tembusu virus (TMUV), a member in the family Flaviviridae, induces egg-drop syndrome and fatal encephalitis in ducks, and thus threatens severely poultry industry. The development of preventive strategies against TMUV infection may contribute significantly to the viral disease control, but specific therapeutics is not currently available for TMUV infection. In this study, a novel TMUV replicon system was constructed in two cell lines by replacing viral structural genes with a Gaussia luciferase (Gluc) reporter and a blasticidin (BSD) resistance marker. Through continuous BSD selection, a stable Vero cell line harboring self-replicating TMUV RNA was established with replication fidelity over at least ten serial passages. Further whole-genome sequencing revealed that two adaptive mutations (C3108T and G5534A), causing NS1(L217L) and NS3(M315I) substitutions, occurred in passages, which resulted in synergistically enhanced TMUV RNA replication in Vero cells. Using the Vero-TMUV replicon system, three compounds, Panobinostat, Floxuridine, and 5-Fluorouracil were screened out as promising antiviral candidates, along with the known flavivirus inhibitor, Lycorine. The development of this stable replicon cell line for TMUV cluster 3 may provide a versatile platform for screening antiviral drugs and also for understanding the host-virus interaction.