Limited potential for mosquito transmission of genetically engineered, live-attenuated Venezuelan equine encephalitis virus vaccine candidates

In an attempt to improve the current live-attenuated Vaccine (TC-83) for Ve nezuelan equine encephalitis (VEE), specific mutations associated with atte nuation of VEE virus in rodent models were identified. These mutations were inserted into full-length cDNA clones of the Trinidad donkey strain of VEE virus by site-directed mutagenesis, and isogenic virus strains with these mutations were recovered after transfection of baby hamster kidney cells wi th infectious RNA. We evaluated 10 of these strains for their ability to re plicate in and be transmitted by Aedes taeniorhynchus, a natural vector of epizootic VEE virus. Two vaccine candidates, one containing a deletion of t he PE2 furin cleavage site, the other a combination of three separate point mutations in the E2 glycoprotein, replicated in mosquitoes and were transm itted to hamsters significantly less efficiently than was either parental ( wild type) VEE virus or TC-83 virus. Although the attenuated strains were t ransmitted to hamsters by mosquitoes, after intrathoracic inoculation, ther e was no evidence of reversion to a virulent phenotype. The mutations that resulted in less efficient replication in, or transmission by, mosquitoes s hould enhance vaccine safety and reduce the possibility of environmental sp read to unintentional hosts.