Development of reverse transcription-PCR assays specific for detection of equine encephalitis viruses

Specific and sensitive reverse transcription-PCR (RT-PCR) assays were devel oped for the detection of eastern, western, and Venezuelan equine encephali tis viruses (EEE, WEE, and VEE, respectively). Tests for specificity includ ed all known alphavirus species, The EEE-specific RT-PCR amplified a 464-bp region of the E2 gene exclusively from 10 different EEE strains from South and North America with a sensitivity of about 3,000 RNA molecules. In a su bsequent nested PCR, the specificity was confirmed by the amplification of a 262-bp fragment, increasing the sensitivity of this assay to approximatel y 30 RNA molecules. The RT-PCR for WEE amplified a fragment of 354 bp from as few as 2,000 RNA molecules. Babanki virus, as well as Mucambo and Pixuna viruses (VEE subtypes IIIA and rv), were also amplified. However, the latt er viruses showed slightly smaller fragments of about 290 and 310 bp, respe ctively. A subsequent seminested PCR amplified a 195-bp fragment only from the 10 tested strains of WEE from North and South America, rendering this a ssay virus specific and increasing its sensitivity to approximately 20 RNA molecules. Because the 12 VEE subtypes showed too much divergence in their 26S RNA nucleotide sequences to detect all of them by the use of nondegener ate primers, this assay was confined to the medically important and closely related VEE subtypes WE, IC, ID, IE, and II. The RT-PCR-seminested PCR com bination specifically amplified 342- and 194-bp fragments of the region cov ering the 6K gene in VEE. The sensitivity was 20 RNA molecules for subtype LAB virus and 70 RNA molecules for subtype IE virus. In addition to the sub types mentioned above, three of the enzootic VEE (subtypes IIIB, IIIC, and IV) showed the specific amplicon in the seminested PCR, The practicability of the latter assay was tested with human sera gathered as part of the febr ile illness surveillance in the Amazon River Basin of Peru near the city of Iquitos, All of the nine tested VEE-positive sera showed the expected 194- bp amplicon of the VEE-specific RT-PCR-seminested PCR.