RESUMEN
Targeting regions of proteins that show a high degree of structural conservation has been proposed as a method of developing immunotherapies and vaccines that may bypass the wide genetic variability of RNA viruses. Despite several attempts, a vaccine that protects evenly against the four circulating Dengue virus (DV) serotypes remains elusive. To find critical conserved amino acids in dengue viruses, 120 complete genomes of each serotype were selected at random and used to calculate conservation scores for nucleotide and amino acid sequences. The identified peptide sequences were analysed for their structural conservation and localisation using crystallographic data. The longest, surface exposed, highly conserved peptide of Envelope protein was found to correspond to amino acid residues 250 to 270. Mutation of this peptide in DV1 was lethal, since no replication of the mutant virus was detected in human cells. Antibodies against this peptide were detected in DV naturally infected patients indicating its potential antigenicity. Hence, this study has identified a highly conserved, critical peptide in DV that is a target of antibodies in infected humans.
Asunto(s)
Virus del Dengue/genética , Virus del Dengue/inmunología , Dengue/inmunología , Péptidos/inmunología , Proteínas del Envoltorio Viral/genética , Secuencia de Aminoácidos , Anticuerpos Antivirales/metabolismo , Secuencia de Bases , Secuencia Conservada , Cristalografía por Rayos X , Dengue/virología , Genoma Viral , Humanos , Modelos Moleculares , Mutación , Péptidos/química , Péptidos/genética , Conformación Proteica , Serogrupo , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
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(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(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(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.
Asunto(s)
Virus del Dengue/patogenicidad , Proteínas del Envoltorio Viral/metabolismo , Proteínas no Estructurales Virales/metabolismo , Factores de Virulencia/metabolismo , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Línea Celular , Culicidae , Virus del Dengue/genética , Modelos Animales de Enfermedad , Humanos , Ratones , Datos de Secuencia Molecular , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , ARN Helicasas/genética , ARN Helicasas/metabolismo , Genética Inversa , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Análisis de Supervivencia , Proteínas del Envoltorio Viral/genética , Proteínas no Estructurales Virales/genética , Virulencia , Factores de Virulencia/genética , Replicación ViralRESUMEN
Recent observations indicate that the clinical profile of dengue virus (DENV) infection is changing, and that neurological manifestations are becoming frequent. The neuro pathogenesis of dengue, and the contribution of viral and host factors to the disease are not well understood. To define the amino acid substitutions in DENV potentially implicated in the acquisition of a neurovirulent phenotype we used a murine model to characterize two neuroadapted strains of DENV-1, FGA/NA a5c (previously obtained), and FGA/NA P6 (recently obtained). Only three amino acid substitutions were identified in the neurovirulent strains, mapping to the E and NS3 helicase domains. These mutations enhanced the ability of neuroadapted viral strains to replicate in the CNS of infected mice, causing extensive damage with leptomeningitis and encephalitis.
