Long-term passage of duck Tembusu virus in BHK-21 cells generates a completely attenuated and immunogenic population with increased genetic diversity

•A completely attenuated viral population was produced by long-term passaging of duck TMUV in BHK-21 cells.•A single-dose vaccination of JXSP-P310 can provide efficient protection for ducks against a virulent TMUV challenge.•Multiple gene mutations co-contribute to the complete attenuation of TMUV.•JXSP-310P is a promising live vaccine candidate against duck TMUV infection. Duck Tembusu virus (TMUV) is an emerging pathogenic flavivirus that causes severe egg-drop and fatal encephalitis in domestic ducks and geese. Although a live-attenuated virus vaccine is effective for disease control, the stability of the attenuation has not been clearly evaluated due to a poor understanding of the attenuation mechanism. Here, a virulent duck TMUV isolate was successively passaged in BHK-21 cells, leading to an approximately 100-fold increase of virus production in cell culture and a complete attenuation of virulence for ducks. The passaged virus induced high titers of TMUV-specific antibody and provided efficient protection against a virulent TMUV challenge after a single-dose vaccination. One hundred and two, and eighteen single-nucleotide polymorphisms (SNPs) at a frequency of >1% were respectively identified in the attenuated virus population and the original isolate by deep sequencing. The increased SNPs numbers suggested that the accumulated variants of virus population may have conferred the phenotypic changes. We cloned and characterized a dominant variant exhibiting similar fitness to the mixed population, and 23 amino acid substitutions were identified across the viral open reading frame. Using reverse genetics, two chimeric viruses were generated by introducing the mutated E or NS5 gene into the backbone of virulent TMUV. We found that mutations in the E gene conferred a fitness advantage in BHK-21 cells and decreased the virus pathogenicity, whereas NS5 mutations reduced the virus infectivity in ducklings without altering the in vitro fitness. In conclusion, increased mutations in a virulent TMUV strain did substantially reduce the virus virulence, and mutations in multiple genes co-contribute to TMUV attenuation.