Chimeric
cDNA clones, pMWE1000 and pMWE2000, differing by five
nucleotides at their 5' termini, were constructed of the 5' two-thirds of the
western equine encephalitis (WEE) virus genome (encoding nonstructural
proteins) and the 3' one-third of the
eastern equine encephalitis (EEE) virus genome (encoding structural
proteins). The WEE virus sequences were derived from full-length
cDNA clones, pWE1000 and pWE2000, which were isogenic except for five
nucleotide differences at their 5' termini and were responsible for significant differences in mouse virulence. Each
cDNA clone was placed downstream from a T7 promoter to allow in vitro transcription of full-length
RNA. Transfection of BHK-21 cells with the chimeric
RNA by electroporation gave rise to high-titer infectious virus. The in vitro characteristics of each chimera virus were determined by electrophoretic analysis of its structural
proteins, plaque morphology, neutralization characteristics, replication kinetics, and rate of
viral RNA synthesis. With the exception of plaque morphology, the in vitro characteristics of MWE1000 and MWE2000 were indistinguishable from the parental EEE virus. Subcutaneous inoculation of 5-week-old C57BL/6 mice with varying doses of MWE1000 or MWE2000 virus demonstrated that both chimeric viruses were significantly attenuated compared to the parental WEE virus (Cba 87) and EEE virus (PE-6). Animals infected with 10(5) PFU or more of either MWE1000 or MWE2000 were completely protected from lethal challenge with the virulent EEE virus, FL91-4679, but were not protected from virulent WEE virus Cba 87 challenge. Construction of viable virus chimeras often results in attenuated viruses that may hold promise as genetically engineered alphavirus
vaccine candidates (R. J. Kuhn, D. E. Griffin, K. E. Owen, H. G. M. Niesters, and J. H. Strauss, 1996, J. Virol. 70, 7900-7909).