The dengue virus family contains 4 distinct serotypes that are distinguishable by virus neutralization. Among the dengue group the type 2 virus is most frequently involved in hemorrhagic fever, a severe and often fatal form of dengue disease. There is considerable polymorphism among type 2 viruses as indicated by variation in oligonucleotide fingerprints and this variation has a geographic distribution in that specific patterns are limited to specific localities. Efforts are now underway to study genetic variation of dengue viruses by molecular cloning and nucleotide sequencing. Dengue 2 (strain PR159) was chosen for sequence comparison with dengue 4 that is also being cloned and sequenced in our laboratory. Dengue 2 genomic RNA was purified and transcribed into RNA-cDNA hybrids for direct cloning in the pBR322 vector according to the procedure established for dengue 4. Analysis of plasmid DNA after Pst I digestion on agarose gel showed that a majority of recombinant plasmids contained DNA inserts ranging from 500-4,000 base pairs in length. Recombinants with the largest inserts (2,000 base-pairs or more) were chosen for mapping the full-length genomic sequence. For this purpose we took advantage of the genetic homology that exists between dengue virus type 2 and type 4. Radiolabeled probes prepared from cloned DNA segments of dengue 4 at various map positions were used for initial mapping and screening of dengue 2 inserts. Thus far, more than one-half of the dengue 2 genomic sequences have been cloned and our ultimate goal is to obtain a full-length DNA copy. Complete sequence analysis will be performed in an effort to gain a better understanding of the pattern of virus polymorphism in dengue epidemics and the involvement of different virus strains in dengue hemorrhagic fever.
