Serum neutralizing antibody was determined using an 80% plaque-reduction neutralization test. Serial fourfold dilutions of serum were prepared in HBSS-FBS. An equal volume of the ZH501 strain of RVFV suspension containing approximately 80 PFU/50 ml was added to each dilution. After incubation at 37uC for 1 hr, 50 ml of each dilution was adsorbed on duplicate Vero cell monolayers for 1 hr at 37uC and then overlaid with 0.6 ml of the agarose-medium mixture used in the viral plaque assay. After 72 hr incubation at 37uC in a 5% CO 2 atmosphere, each monolayer received 0.6 ml of a second agarose containing neutral red dye. Plaques were counted and an 80% reduction in the number of plaques inoculated was used as the endpoint for virusneutralization titers.