The main objectives of this research are; 91) identify viral factors which may influence the severity of dengue infection in humans, by comparing specific viral genome differences with clinical and epidemiologic observations, and (20 to correlate these genome differences with human monocyte infection rates, a possible in vitro marker for human virulence. We will determine the present distribution of dengue virus genotypes, of serotype 2, in Bangkok, by phylogenetic analysis of 20 isolates, using limited sequencing techniques (comparison of 240 nucleotides from the E/NS1 gene junction). This study will provide preliminary data on disease associations with specific genotypes, as viruses are obtained from project 1. Experiments will be carried out to determine the feasibility of using the polymerase chain reaction (PCR) for the amplification of dengue virus RNA, for sequencing. We will determine if there is a correlation between disease severity and specific nucleotide differences in the virus genome, by comparing the complete genomic sequences of 6 dengue viruses of serotype 2 (3 pairs from matched D and DHF patients). These viruses will be selected from patients in one location, with complete clinical and immunologic workups (from projects 1 and 2), to assign them to each of two categories, D or DHF, and by whether the viruses replicate to high or low levels in human monocytes, a possible in vitro model of virulence. We will confirm and correlate significant genetic differences seen in the above mentioned viruses with those of 20 additional viruses from each of the two clinical categories (D and DHF), by sequencing the implicated areas of the genome. These molecular virology data will be combined with the detailed immunologic data (from Projects 1 and 2), to develop an improved model for interactions of human immune responses and dengue viral determinants that result in the production of severe dengue disease, and will provide data that will be useful in the development of dengue vaccine.
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