Molecular Cloning and Sequencing of the 26S Region of Western Equine EncephalitisVirus Strain 71V-1658

摘要

A 3100 bp cDNA clone of the 26S region of western equine encephalitis (WEE) virusstrain 71V-1658 was identified by dot blot hybridization from a cDNA library. The missing 5' end was PCR-amplified and engineered into pcDW-12 to obtained a full length clone of the 26S region. The resulting construct (XH-7) was restriction mapped and completely sequenced on both strands. Only eleven of the sixty-three nucleotide differences resulted in amino acid changes when the sequence was compared to WEE strain BFS-1703. In addition, the high degree of conservation of the structural proteins was maintained when compared to the N-terminal sequence of the El and E2 proteins of the McMillan strain of WEE. The conserved nature of the structural proteins would indicate that one strain of WEE should be able to cross-protect against all WEE strains. A 2.2 kb fragment at the 5' end of the genome was also cloned and sequenced, and demonstrated high homology to the available sequences for eastern equine encephalitis (EEE) and Highlands J (HJ) viruses, adding further evidence that the entire 5' nonstructural region of WEE was derived from EEE. In summary, the genetic cloning and sequencing of the WEE 26S region marks the critical first step in the generation of a subunit vaccine to WEE.