DENV-3 GENÓTIPO I L1: o que determina a neurovirulência?

Abstract

Dengue is a febrile, acute and systemic disease with a broad clinical spectrum, which can range from asymptomatic to severe cases and death. In a hypernedemic area, symptoms previously considered rare are becoming more frequently opposing the benign course disease idea. Since the first identification of dengue virus 3 genotype 1 strain 1 (DENV-3 GI L1) in Minas Gerais (MG) by our research group, an association of this virus has been observed in the development of severe cases. Several studies involving epidemiology, molecular biology, immunological mediators, in vitro and in vivo analyses have been done, indicating a relationship between this lineage and neurological symptoms. DENV-3 MG-20 was isolated in 2003 from the serum of a lethal case with neurological symptoms and was used as a neurovirulent prototype sample in this study. During the epidemiological survey to verify incidence, distribution, and symptomatology, we identified reports that confirm our suspicions in the association of this genotype with neurovirulence in different parts of the world. However, a Rondonia (RO) group DENV-3 isolates with high identity to MG viruses paradoxically were non-neurovirulent in mice models. Phylogenetic analysis identified molecular markers in the envelope protein region (E). One of the isolates from the same region and markers was analyzed for neurovirulence in mice. The DENV-3 PV_BR was inoculated in BALB / C mice compared to DENV-3 MG-20, showing no clinical signs. The protein E sequences of both viruses were analyzed by comparative modeling, showing that amino acids (aa) 62 and 123 were in very close regions, possibly participating in the same biological interaction. These aa have been related to sites of interaction with sulfated glycosaminoglycans-GAGs (especially heparin / heparan sulfate, analogous molecules). Similarly, alteration of GAG sensitivity has been largely associated with neurovirulence alterations of neurotropic flaviviruses. The E62K, E123Q substitutions for DENV-3 MG-20 and DENV-3 PV_BR respectively are non-conservative, including charge reversal, which generated a high impact on in silico electrostatic density and heparin molecule docking analyzes. In vitro tests with heparin and heparinases indicated the biological impact of these changes.Other characteristics previously associated with changes in cell tropism and neurovirulence, such as changes in the size of lysis plaques and differences in cytopathic effect (ECP) were also observed. ECP differences in C6 / 36 mosquito cells and human glioblastoma U251 cells were observed, in which U251 cells were destroyed only by infection with DENV-3 MG-20. Immunofluorescence (IFI) analyzes showed multiple syncytia formation before lysis of DENV-3 MG-20 and the virus was also identified by transmission electron microscopy. Due to the importance of protein E in host cell interaction, in addition to the compatibility of in silico analyses, characterization, and previous descriptions, it is highly probable that the E62K, E123Q substitutions are responsible for the alteration in neurovirulence in mice. Although this mechanism is multifactorial, interaction with GAGs seems to be a strong component in neurovirulence.