Genetic Determinants Responsible for Acquisition of Dengue Type 2 Virus Mouse Neurovirulence

Abstract

Studies conducted some 50 years ago showed that serial intracerebral passage of dengue viruses in mice selected for neurovirulent mutants that also exhibited significant attenuation for humans. We investigated the genetic basis of mouse neurovirulence of dengue virus because it might be directly or indirectly associated with attenuation for humans. Analysis of the sequence in the C-PreM-E-NS1 region of the parental dengue type 2 virus (DEN2) New Guinea C (NGC) strain and its mouse-adapted, neurovirulent mutant revealed that 10 nucleotide changes occurred during serial passage in mice. Seven of these changes resulted in amino acid substitutions, i.e., Leu 55 -Phe and Arg 57 -Lys in PreM, Glu 71 -Asp, Glu 126 -Lys, Phe 402 -Ile, and Thr 454 -Ile in E, and Arg 105 -Gln in NS1. The sequence of C was fully conserved between the parental and mutant DEN2. We constructed intertypic chimeric dengue viruses that contained the PreM-E genes or only the NS1 gene of neurovirulent DEN2 NGC substituting for the corresponding genes of DEN4. The DEN2 (PreM-E)/DEN4 chimera was neurovirulent for mice, whereas DEN2 (NS1)/DEN4 was not. The mutations present in the neurovirulent DEN2 PreM-E genes were then substituted singly or in combination into the sequence of the nonneurovirulent, parental DEN2. Intracerebral titration of the various mutant chimeras so produced identified two amino acid changes, namely, Glu 71 -Asp and Glu 126 -Lys, in DEN2 E as being responsible for mouse neurovirulence. The conservative amino acid change of Glu 71 -Asp probably had a minor effect, if any. The Glu 126 -Lys substitution in DEN2 E, representing a change from a negatively charged amino acid to a positively charged amino acid, most likely plays an important role in conferring mouse neurovirulence.