On the basis of nucleotide sequencing and serologic tests, including plaque reduction neutralization tests, 4 major lineages/clusters (clusters 1-4) with distinct antigenic subtypes have been identified (Brault et al., 1999) . Serial dilutions were made of the RNA from representatives of each cluster (Table 3) . We then performed the singleplex real-time RT-PCR assay for the detection of EEEV on various RNA dilutions to determine whether nucleic acid from the 12 different strains would be amplified by the real-time RT-PCR reaction.