COUPLED PCR-RESTRICTION ENZYME ANALYSIS FOR RAPID IDENTIFICATION OF STRUCTURAL GENE RELATIONSHIPS AMONG STRAINS OF EASTERN EQUINE ENCEPHALITIS VIRUS

摘要

We have used restriction endonuclease digestion analysis of polymerase chain reaction (PCR)-amplified gene regions to rapidly examine individual structural gene relationships among field isolates of eastern equine encephalitis (EEE) virus. The E1(+) (E1 gene plus 292 nucleotides 3' of the coding region), E2, and C gene regions from North American (NA) variety viruses and the E1 and C gene regions of South American (SA) variety viruses were successfully amplified by RT-PCR using a single primer set for each locus. The products were then digested with a panel of restriction endonucleases and the resulting DNA fragments electrophoretically compared. Our findings revealed marked similarity among the E1(+) and the E2 gene restriction patterns, respectively, of most NA strains. In contrast, the restriction patterns exhibited by the E1(+) gene of SA strains differed substantially from those of NA strains and also appeared more heterogeneous. The digestion patterns of the C gene were generally similar for all strains of the virus examined. These results thus demonstrate that EEE viral E1(+) and C structural gene sequences can be amplified from an assortment of both NA and SA varieties of the virus by RT-PCR using a single primer set per locus, and that both varietal and individual isolate distinctions can be identified by comparison of subsequent restriction digestion patterns. This technique should prove useful as an epidemiological tool for rapid identification of EEE isolates from clinical and field specimens, and as a rapid screen for alterations within structural gene regions.