Construction of intertypic chimeric dengue viruses exhibiting type 3 antigenicity and neurovirulence for mice

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.