A genomic-length cDNA clone corresponding to the RNA of dengue virus type 2 (DEN-2) New Guinea C strain (NGC) was constructed in a low copy number vector. The cloned cDNA was stably propagated in Escherichia coil and designated pDVWS501. RNA transcripts produced in vitro from the cDNA using T7 RNA polymerase yielded infectious virus (MON501) upon electroporation into BHK-21 cells. When compared with parental NGC virus, MON501 replicated to similar levels in Aedes albopictus C6/36 cells and showed similar neurovirulence in suckling mice. In contrast, a second genomic-length cDNA clone (pDVWS310) used as an intermediate in the construction of pDVWS501 produced virus (MON310) that replicated well in C6/36 cells but was not neurovirulent in mice. MON310 contained the prM and E genes of the non-neurovirulent PUO-218 strain in an NGC background. There were seven amino acid differences between the prM and E proteins of MON310 and MON501. The differences were generally conservative, with the exception of E residue 126, which was Glu in MON310 and Lys in MON501. To examine the role of this residue in mouse neurovirulence, substitutions of Glu --> Lys and Lys --> Glu were made in MON310 and MON501, respectively. The properties of these mutants clearly demonstrated that Lys at E residue 126 is a major determinant of DEN-2 mouse neurovirulence.


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