Mouse-adapted dengue type 4 virus (DEN4) strain H241 is highly neurovirulent for mice, whereas its non-mouse-adapted parent is rarely neurovirulent. The genetic basis for the neurovirulence of the mouse-adapted mutant was studied by comparing intratypic chimeric viruses that contained the three structural protein genes from the parental virus or the neurovirulent mutant in the background sequence of nonneurovirulent DEN4 strain 814669. The chimera that contained the three structural protein genes from mouse neurovirulent DEN4 strain H241 proved to be highly neurovirulent in mice, whereas the chimera that contained the corresponding genes from its non-mouse-adapted parent was not neurovirulent. This finding indicates that most of the genetic loci for the neurovirulence of the DEN4 mutant lie within the structural protein genes. A comparison of the amino acid sequences of the parent and its mouse neurovirulent mutant proteins revealed that there were only five amino acid differences in the structural protein region, and three of these were located in the envelope (E) glycoprotein. Analysis of chimeras which contained one or two of the variant amino acids of the mutant E sequence substituting for the corresponding sequence of the parental virus identified two of these amino acid changes as important determinants of mouse neurovirulence. First, the single substitution of Ile for Thr-155 which ablated one of the two conserved glycosylation sites in parental E yielded a virus that was almost as neurovirulent as the mouse-adapted mutant. Thus, the loss of an E glycosylation site appears to play a role in DEN4 neurovirulence. Second, the substitution of Leu for Phe-401 also yielded a neurovirulent virus, but it was less neurovirulent than the glycosylation mutant. These findings indicate that at least two of the genetic loci responsible for DEN4 mouse neurovirulence map within the structural protein genes.

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There are four dengue virus serotypes (DEN1 to -4), each of which causes major epidemics in tropical or subtropical areas. The current strategy for dengue virus immunization favors the use of a tetravalent vaccine preparation. We have previously employed full-length DEN4 cDNA to construct a viable intertypic dengue virus type 1 or type 2 chimera that contained the C-PreM-E or only the PreM-E genes of DEN1 or DEN2 substituting for the corresponding genes of DEN4. This success implied that it might be possible to create mutants of all four dengue virus serotypes for evaluation as candidate vaccines. In this study, we constructed DEN3-DEN4 chimeras that contained DEN3 C-PreM-E genes and expressed DEN3 antigenic specificity. Unlike our previous successes in cloning DEN1 or DEN2 chimeric cDNA, we were not able to clone the DEN3 C-PreM-E genes directly in the 5' intermediate vector or in the full-length chimeric DEN3-DEN4 plasmid in Escherichia coli. Nevertheless, a full-length DNA template of DEN3-DEN4 that could be used for transcription of infections RNAs was prepared by in vitro ligation. Progeny virus recovered from RNA-transfected C6/36 mosquito cells exhibited DEN3 antigenic specificity as determined by a reaction with monoclonal antibodies. Gel electrophoresis of virus-infected cell lysates yielded the predicted viral protein pattern, i.e., DEN3 C, PreM, and E and DEN4 nonstructural proteins. Two amino acid substitutions, Thr-435-->Leu and Glu-406-->Lys, which are analogous to mutations that, respectively, confer mouse neurovirulence on DEN4 and DEN2, were introduced into DEN3 E. A mutant chimera containing the Thr-435-->Leu substitution, which ablates the potential glycosylation site sequence, produced an E protein identical in size to that of wild-type DEN3 E, indicating that the glycosylation site is normally not used. Intracerebral inoculation of suckling mice revealed that the mutant chimera containing the Glu-406-->Lys substitution was neurovirulent, whereas its wild-type counterpart or parent DEN3 was not.

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