La entrada de virus dengue a líneas celulares humanas en la infección primaria en ausencia o presencia de anticuerpos

Abstract

At present, a high proportion of world population is at risk of infection with dengue virus (DENV), a pathogen causing either a mild febrile illness or more severe forms of disease, with 2.5 % mortality. Although DENV represents a serious health problem worldwide, no specific chemotherapy or vaccine is currently available. Virus entry is an attractive antiviral strategy to block initiation of infection. For DENV, the mode of entry into the host cell has been studied only in fibroblastic or epithelial cell lines, derived from mammals or mosquitoes, with controversial results. In the present study, the entry of DENV into human myeloid cell lines, a model more representative of the natural infection, was analyzed as well as the relationship between mode of entry and antiviral susceptibility to sulfated polysaccharides. By using biochemical inhibitors of endocytic routes, infectivity titrations by plaque formation, viral RNA determinations by quantitative RT/PCR, fluorescence and electron microscopy, a clathrin, and dynamin, mediated endocytosis was demonstrated for entry of both serotypes DENV 2 strain NGC and DENV 1 strain Hawaii into human myelomonocytic U937 and erithroleukaemic K562 cell lines. When both cells were infected with DENV 2 in the presence of nonneutralizing anti DENV antibodies, conditions which allow an enhancement of infection in vitro and lead to severe forms of disease in vivo, the route of entry was clathrin mediated or not, according to the receptor FcR involved in the infective process. Furthermore, the antiviral activity in both cell systems of carrageenan, a sulfated polysaccharide known to inhibit DENV 2 adsorption and uncoating in Vero cells, was also evaluated. A relationship between antiviral susceptibility to carrageenan and the type of receptor employed for antibody-mediated entry was observed. Finally, studies about the cholesterol dependence for DENV infection have shown that the envelope cholesterol is a critical factor in the fusion process for DENV entry whereas cell membrane cholesterol is not involved. The characterization of the mode of entry in myeloid cells and the role of viral cholesterol contributes to the knowledge of factors involved in DENV infection and the design and usage of new and more effective antiviral therapies.