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The nucleotide sequences of the envelope genes from five Thai and three Sri Lankan dengue-2 viruses were determined by sequencing the viral RNA using synthetic oligonucleotide primers. The results were compared with the four published dengue-2 envelope sequences to obtain a classification of these viruses, which showed that the Thai isolates could be divided into two separate groups while the Sri Lankan isolates were distinct. There was no correlation between disease severity and envelope protein sequence, or between year of isolation and sequence. No particular amino acid changes were associated with virulence or a change in hydrophilic region which could perhaps act as an epitope.

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SUMMARYPrevious studies have compared RNA genomes from the different dengue virus serotypes by cDNA-RNA hybridization using dengue-1 virus-and dengue-2 virusspecific cDNA probes. These probes revealed that there is a close genetic relationship between dengue virus serotypes 1 and 4. In this communication, the cDNA-RNA hybridization results using dengue-3-and dengue-4-specific cDNA probes to determine the genetic relatedness of all four dengue virus serotypes are reported. The results indicate that serotypes 1 and 4 are genetically very closely related (sharing about 70% of their genomes as detected by both the dengue-1 and dengue-4 CDNA probes), as are serotypes 3 and 4 (sharing about 50~o of their genomes as detected by both the dengue-3 and dengue-4 cDNA probes). Serotype 2 does not seem to be very closely related to the other dengue virus serotypes by cDNA-RNA hybridization analysis.Flaviviruses are a group of related viruses within the family Togaviridae, which replicate in arthropod and vertebrate cells. Some of these viruses cause major diseases such as Japanese encephalitis, yellow fever and dengue haemorrhagic fever in humans. Flaviviruses are subdivided by their reactions in serological tests. Each virus contains antigenic determinants common to the group, others which specify a complex of viruses and those which determine serotype specificity (Trent, t977). There are four dengue virus serotypes which form a discrete complex of flaviviruses (Porterfield, 1980), and each of these serotypes is associated with a wide range of clinical manifestations.Dengue viruses consist of a single-stranded RNA genome (about 12000 nucleotides long), a capsid protein (C), a low mol. wt. membrane protein (M) and an envelope glycoprotein (E) (for review, see Schlesinger, 1977). To determine the relatedness of dengue virus RNA genomes from each of the four serotypes, near full-length serotype-specific cDNA probes were synthesized and used in cDNA-RNA hybridization experiments. Studies with dengue-l-and dengue-2-specific cDNA probes were reported previously (Blok et al., 1984) and results with dengue-3-and dengue-4-specific probes are described below.Prototype viruses of the four dengue serotypes (dengue-l, Haw; dengue-2, NGC; dengue-3, H87; dengue-4, H241) were grown in the C6/36 clone of Aedes albopictus cells, and virus particles extruded into the medium were purified by polyethylene glycol precipitation. Viral RNA was extracted from the virus pellets and used either as a template for cDNA synthesis in vitro using an oligo(dT) primer and the enzyme reverse transcriptase, or in cDNA-RNA hybridization experiments as described earlier (Blok et al., 1984).In order to synthesize the amount of radioactively labelled cDNA needed for cDNA-RNA hybridization analyses, the concentrations of dengue-3-and dengue-4-specific RNA, of oligo (dT)12_18 primer, [32p]dCTP and avian myeloblastosis virus reverse transcriptase were increased tenfold over that used in previous studies with RNA from dengue-1 and dengue-2 serotypes (Blok et al., 198...