Characterization of infectious Murray Valley encephalitis virus derived from a stably cloned genome-length cDNA

An infectious cDNA clone of Murray Valley encephalitis virus prototype stra in 1-51 (MVE-1-51) was constructed by stably inserting genome-length cDNA i nto the low-copy-number plasmid vector pMC18. Designated pMVE-1-51,the clon e consisted of genome-length cDNA of MVE-1-51 under the control of a T7 RNA polymerase promoter. The clone was constructed by using existing component s of a cDNA library, in addition to cDNA of the 3' terminus derived by RT-P CR of poly(A)tailed viral RNA. Upon comparison with other flavivirus sequen ces, the previously undetermined sequence of the 3' UTR was found to contai n elements conserved throughout the genus Flavivirus. RNA transcribed from pMVE-1-51 and subsequently transfected into BHK-21 cells generated infectio us virus. The plaque morphology, replication kinetics and antigenic profile of clone-derived virus (CDV-1-51) was similar to the parental virus in vit ro, Furthermore, the virulence properties of CDV-1-51 and MVE-1-51 (LD50 va lues and mortality profiles) were found to be identical in vivo in the mous e model. Through site-directed mutagenesis, the infectious clone should ser ve as a valuable tool for investigating the molecular determinants of virul ence in MVE virus.