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., Leu55-Phe and Arg57-Lys in PreM, Glu71-Asp, Glu126-Lys, Phe402-Ile, and Thr454-Ile in E, and Arg105-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, Glu71-Asp and Glu126-Lys, in DEN2 E as being responsible for mouse neurovirulence. The conservative amino acid change of Gu71-Asp probably had a minor effect, if any. The Glu126-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.

FIG. 1

Plasmid constructs of chimeric DEN2 (PreM-E)/DEN4 viruses and analysis of neurovirulence in suckling mice. Chimeric cDNAs that contained the PreM-E genes of DEN2-P or DEN2-N, or their derived sequences, were constructed by using the PstI site previously introduced at the end of the DEN4 C gene for joining with the DEN2 DNA fragment cleaved at the PstI site (nucleotide [nt] 400) and the XhoI site near the end of the DEN2 E sequence. A new MluI site was introduced at nt 888, generating a conservative Ala₂₆₆-Val substitution at the hydrophobic N terminus of E. In addition, two BamHI sites at nt 1696 and 2203 were also chosen to generate DEN2-N PreM-E DNA subfragments to substitute for the corresponding DEN2-P DNA sequences in the series of chimeras containing amino acid substitutions in the PreM-E polyprotein. The procedure to construct chimeric cDNAs from p5′-2 (XhoI) that contained the cloned DEN2 PreM-E genes and p3′-A that contained most of the remaining DEN4 DNA sequences was essentially as described earlier (4). Transcription of Asp₇₁₈ linearized chimeric cDNA and transfection of mosquito C6/36 cells with the RNA transcripts were done as described previously (20). One week after transfection, the cells were transferred to a T₇₅ flask and also to a chamber slide. An indirect immunofluorescence assay was performed with cells in the chamber slide to monitor the fraction of cells infected with progeny virus. Virus was harvested from the medium fluid, and titers were determined on monolayers of C6/36 cells, when 80% or more of cells showed positive fluorescence. Chimeras containing the PreM-E of DEN2-N or DEN2-P or derived mutants were recovered readily, reaching 10⁶ PFU/ml or more 10 to 12 days after transfection. The initial harvest of the chimera containing DEN2 NS1 was obtained under the same condition. This chimera was subsequently amplified once in C6/36 cells to give a titer of 2 × 10⁷ PFU/ml. The series of intertypic dengue chimeric viruses, the parental nonneurovirulent DEN2, and the mouse-adapted neurovirulent DEN2 mutant were analyzed for neurovirulence in mice by intracerebral inoculation (4, 18).

FIG. 2

Analysis of dengue virus proteins by radioimmunoprecipitation. Confluent mosquito C6/36 cells in a T₂₅ flask were infected with the chimera (DEN2 (NS1)/DEN4 [(D2 NS1)/D4] or with DEN2-N or the DEN4 control at a multiplicity of 1.0 for 6 days. Infected cells were then labeled with [³⁵S]methionine (specific activity, 3,000 Ci/mmol; 100 μCi/flask in 2 ml of methionine-free minimal essential medium) for 6 h. The cells were then lysed with RIPA buffer (1% sodium deoxycholate, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate [SDS], 0.01 M Tris-HCl [pH 7.5], 0.15 M NaCl), and the radiolabeled lysates were analyzed by immunoprecipitation with DEN2 (lanes 2)- or DEN4 (lanes 4)-specific hyperimmune mouse ascitic fluid or with DEN2 NS1-specific monoclonal antibody 34-23 (lanes M). The immunoprecipitates were analyzed by SDS-polyacrylamide gel electrophoresis. The radiolabeled protein bands were visualized by autoradiography of the dried gel.

FIG. 3

Mouse neurovirulence of DEN2 (NS1)/DEN4. Three-day-old Swiss outbred mice in groups of 10 were inoculated intracerebrally with the chimera DEN2 (NS1)/DEN4 at 1,000 PFU. Neurovirulent DEN2-N, nonneurovirulent DEN4, and the derived DEN2 (C-PreM-E)/DEN4 chimera at the same dose were included as controls. Inoculated mice were observed daily for signs of encephalitis, and deaths were recorded for a 24-day period postinoculation.