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The Tembusu virus (TMUV) PS strain, derived by several passages and plaque purifications in BHK-21 cells, displays markedly lower virulence in Pekin ducklings relative to a natural isolate of TMUV, but the potential virulence determinants and the in vivo mechanisms for substantial virulence attenuation of the passage variant remain unknown. Here, we constructed a series of chimeric and mutant viruses and assessed their virulence using a 2-day-old Pekin duckling model. We showed that residue 304 in the envelope (E) protein is the molecular determinant of TMUV virulence. Further investigations with mutant and parental viruses demonstrated that acquisition of positive charges at E protein residue 304 plays a critical role in substantial attenuation of neurovirulence and neuroinvasiveness, which is linked to enhanced binding affinity for glycosaminoglycans (GAGs). In Pekin ducklings infected by subcutaneous inoculation, an Arg at residue 304 in the E protein was shown to contribute to more rapid virus clearance from the circulation, markedly reduced viremia, and significantly decreased viral growth in the extraneural tissues and the central nervous system, relative to a Met at the corresponding residue. These findings suggest that the in vivo mechanism of virulence attenuation of the TMUV passage variant closely resembles that proposed previously for GAG-binding variants of other flaviviruses. Overall, our study provides insight into the molecular basis of TMUV virulence and the in vivo consequences of acquisition of a GAG-binding determinant at residue 304 in the E protein of TMUV. [ABSTRACT FROM AUTHOR]