Wild rabies virus detection by plaque assay from naturally infected brains in different species.

A simple, sensitive and specific plaque assay protocol for the detection of wild type rabies virus in different species is described using confluent monolayers of chicken embryo cells in 6-well plates. Plaques are produced after application of either agarose or Sephadex G-100 overlay onto cell monolayers and incubation for 96 h after virus infection at 37 degrees C. The parameters affecting plaque appearance include cell seeding concentration, overlay composition and time of incubation after infection. Optimal conditions are seeding at a concentration of 4 x 10(6) cell/cm(3), incubation at 37 degrees C in 5% CO(2) atmosphere during 96 h, using either 1% agarose or 2% Sephadex G-100 overlays. The described plaque assay would be a new valuable tool in conducting various quantitative investigations, since the chicken embryo cells are susceptible to rabies virus infection from all species studied.

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 Venezuelan equine encephalitis (VEE), specific mutations associated with attenuation 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 with infectious RNA. We evaluated 10 of these strains for their ability to replicate in and be transmitted by Aedes taeniorhynchus, a natural vector of epizootic VEE virus. Two vaccine candidates, one containing a deletion of the PE2 furin cleavage site, the other a combination of three separate point mutations in the E2 glycoprotein, replicated in mosquitoes and were transmitted to hamsters significantly less efficiently than was either parental (wild type) VEE virus or TC-83 virus. Although the attenuated strains were transmitted to hamsters by mosquitoes, after intrathoracic inoculation, there was no evidence of reversion to a virulent phenotype. The mutations that resulted in less efficient replication in, or transmission by, mosquitoes should enhance vaccine safety and reduce the possibility of environmental spread to unintentional hosts.