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nonstructural proteins NS1, NS3, and NS5 of the New Guinea C strain of dengue virus type 2 (DEN-2) were obtained by nucleotide and protein sequencing. Clones were prepared containing cDNA of DEN-2 virus in the plasmid pUC8. The nucleotide sequences of viral cDNA inserts were determined and the cDNA of each clone positioned on the flavivirus genomic map by comparison of the deduced amino acid sequence with that of yellow fever virus. Radiolabelled E, NS1, NS3 and NS5 were purified by lectin affinity chromatography and preparative gel electrophoresis. Purified proteins were subsequently analysed by Edman degradation to establish the origins of the amino termini of these proteins in the deduced DEN-2 amino acid sequence. Thus the amino acid sequences surrounding the likely proteolytic cleavage sites used in the formation of these four proteins were determined. Of particular interest was the sequence containing the amino terminus of NS3, namely Lys-Lys-Gln-Arg-Ala-Gly where Ala is the first amino acid of NS3. Cleavage following one basic residue in the flavivirus polyprotein has not been reported previously.

Bielefeldt Ohmann, H., D. W. Beasley, et al. (1997). "Analysis of a recombinant dengue-2 virus-dengue-3 virus hybrid envelope protein expressed in a secretory baculovirus system." J.Gen.Virol. 78(Pt 11): 2723-33.
In a step towards a tetravalent dengue virus subunit vaccine which is economical to produce, highly immunogenic and stable, a hybrid dengue virus envelope (E) protein molecule has been constructed. It consists of 36 amino acids from the membrane protein, the N-terminal 288 amino acids of the dengue-2 virus E protein plus amino acids 289-424 of the dengue-3 virus E protein. It has been engineered for secretory expression by fusion to a mellitin secretory signal sequence and truncation of the hydrophobic transmembrane segment. Using the baculovirus expression system and serum-free conditions, more than 95% of recombinant dengue-2 virus-dengue-3 virus hybrid E protein (rD2D3E) was secreted into the cell culture supernatant in a stable form with multiple features indicative of preserved conformation. The hybrid molecule reacted with a panel of dengue virus- and flavivirus-specific MAbs which recognize linear or conformational epitopes on dengue virions. Human dengue virus-specific antisera also reacted with the protein. The hybrid rD2D3E protein was able to inhibit the in vitro binding of dengue-2 and dengue-3 viruses to human myelomonocytic cells, suggesting that the receptor-binding epitope(s) was preserved. Adjuvant-free immunization with the hybrid protein induced an antibody response to both dengue-2 and dengue-3 virus in outbred mice, comparable in strength to that of individual rD2E and rD3E proteins. Notably, these antibody responses were primarily of the IgG2a and IgG2b isotype. A strong dengue virus cross-reactive T cell response was also induced in the mice, suggesting that dengue virus hybrid E proteins could form the basis of an efficacious multivalent dengue virus vaccine.

Bielefeldt Ohmann, H. (1997). "Pathogenesis of dengue virus diseases: missing pieces in the jigsaw." Trends Microbiol. 5(10): 409-13.
The mechanisms involved in the pathogenesis of dengue hemorrhagic fever and dengue shock syndrome remain unresolved. Antibody-dependent enhancement of infection has long been thought to play a central role; however, this remains unverified. The alternative hypothesis that virus variation, virulence and dynamics may account for severe dengue disease, particularly in children, should be considered.