Molecular Strategy for the Construction of a Genetically Engineered Vaccine for Venezuelan Equine Encephalitis Virus.

摘要

The glycoprotein genes of 11 rapidly penetrating, attenuated mutants isolated previously have been sequenced; sequencing of the remaining two mutants is in progress. Five putative attenuating loci have been identified. We have constructed a full-length cDNA clone of TRD-VEE which, when transcribed in vitro, yields an infectious RNA. VEE virus derived from the clone is indistinguishable from TRD-VEE in cell culture and in vivo. Full-length clones containing each of the putative attenuating mutations individually have been constructed using site-directed mutagenesis. Using virus derived from these constructs, we have reproduced the attenuated phenotypes of the biological mutants. Similarly, we have combined the individual attenuating mutations to derive double and triple mutant clones. Virus produced from the single, double and triple mutant constructs tested to date have been avirulent in adult mice and induce protective immunity to challenge with 103.