Synergistic interactions between the [NS3.sub.hel] and E proteins contribute to the virulence of dengue virus type 1

Background: Dengue includes a broad range of symptoms, ranging from fever to hemorrhagic fever and may occasionally have alternative clinical presentations. Many possible viral genetic determinants of the intrinsic virulence of dengue virus (DENV) in the host have been identified, but no conclusive evidence of a correlation between viral genotype and virus transmissibility and pathogenicity has been obtained. Methodology/Principal Findings: We used reverse genetics techniques to engineer DENV-1 viruses with subsets of mutations found in two different neuroadapted derivatives. The mutations were inserted into an infectious clone of DENV-1 not adapted to mice. The replication and viral production capacity of the recombinant viruses were assessed in vitro and in vivo. The results demonstrated that paired mutations in the envelope protein (E) and in the helicase domain of the NS3 ([NS3.sub.hel]) protein had a synergistic effect enhancing viral fitness in human and mosquito derived cell lines. E mutations alone generated no detectable virulence in the mouse model; however, the combination of these mutations with [NS3.sub.hel] mutations, which were mildly virulent on their own, resulted in a highly neurovirulent phenotype. Conclusions/Significance: The generation of recombinant viruses carrying specific E and [NS3.sub.hel] proteins mutations increased viral fitness both in vitro and in vivo by increasing RNA synthesis and viral load (these changes being positively correlated with central nervous system damage), the strength of the immune response and animal mortality. The introduction of only pairs of amino acid substitutions into the genome of a non-mouse adapted DENV-1 strain was sufficient to alter viral fitness substantially. Given current limitations to our understanding of the molecular basis of dengue neuropathogenesis, these results could contribute to the development of attenuated strains for use in vaccinations and provide insights into virus/host interactions and new information about the mechanisms of basic dengue biology.