The scorpion peptide Eval418 inhibits duck Tembusu virus replication by disrupting viral structure and early-stage infection.

Abstract

Introduction Duck Tembusu virus (DTMUV) causes egg-drop syndrome and neurological symptoms in ducks, resulting in significant economic losses. Currently, no specific antiviral drugs are available for DTMUV infection. Scorpion-derived peptides have emerged as promising lead compounds for antiviral drug development. In this study, we investigated the antiviral activity and mechanism of the scorpion peptide Eval418 against DTMUV infection in vitro. Methods DF-1 cells were infected with DTMUV at an MOI of 0.1 and treated with Eval418 (0-20 μM), with Ribavirin as a positive control. Antiviral activity was assessed by Western blot, RT-qPCR, and immunofluorescence assays targeting the DTMUV NS3 protein. Cytotoxicity and hemolytic activity were evaluated in DF-1, DEF, chicken and duck red blood cells the influence of duck serum on Eval418 was examined. Time-of-addition, free virion, attachment, and entry/fusion assays were performed, along with transmission electron microscopy and molecular docking/MD simulations of the NS3-Eval418 complex. Results Eval418 inhibited DTMUV replication in a concentration-dependent manner, achieving over 99% inhibition at 10 μM, with no significant cytotoxicity or hemolytic activity up to 120 μM in all tested cells. Mechanistic studies revealed that Eval418 acted primarily during the coaddition stage by directly inactivating free virions, blocking viral attachment, and inhibiting viral entry/fusion. TEM confirmed that Eval418 directly disrupted the morphological integrity of DTMUV virions, causing structural collapse. MD simulations showed that Eval418 binds stably to the NS3 binding pocket. Eval418 also inhibited two genetically distinct DTMUV isolates, whereas prolonged incubation with duck serum progressively reduced its antiviral activity. Conclusion These findings establish Eval418 as a promising lead compound for the development of antiviral therapeutics against DTMUV infection as a model flavivirus. Its potential efficacy against other flaviviruses requires further investigation.