Estudio de la capacidad antiviral de ribavirina y nano-partículas de plata en células infectadas con bunyavirus mediante técnicas de imagen

Resumen

One of the most important consequences of climate change is the rapid dissemination of the vectors of arboviruses such as Dengue, Zika, Chikungunya and Bunyaviruses. These viruses constitute a global threat because there are currently no vaccines nor specific antiviral drugs for many of them. With fluorescent recombinant bunyaviruses and Correlative Light and Electron Microscopy (CLEM), combined with other virology and cell biology methods, we have studied the response of Vero cells to bunyavirus infection and the effects of two different antivirals, that are Ribavirin (RBV) and Silver Nanoparticles (AgNPs). Bunyamwera virus (BUNV), the prototype of the order Bunyavirales, assembles small replication factories in Vero cells. These structures are made of fragmented Golgi stacks surrounded by mitochondria and Endoplasmic Reticulum (ER) cisternae. With the fluorescent, recombinant virus rBUNGc-eGFP and live cell imaging, it was observed that BUNV particles accumulate in large vacuolar structures that are transmitted from cell to cell. This is a new way of bunyavirus transmission that in Vero cells works together with the reported constitutive secretion of viruses in secretory vesicles. In addition, we observed that BUNV is able to induce drastic changes in the nucleus and nucleolus of Vero cells. Notably, the nucleolus progressively fragments and disappears in BUNV-infected Vero cells, feature that is a genuine hallmark of BUNV infection in Vero cells. Through the use of the mutant rBUN-DelNSs that lacks the non-structural protein (NSs), it was found that NSs is responsible for the disorganization of the nucleus and nucleolus and might also participate in the biogenesis of the viral factory. RBV and AgNPs blocked the infection in ~ 90% of cells. Those cells where the virus escaped from the antiviral treatment, were selected and studied in detail. A small percentage of cells (~3%) presented a peculiar phenotype. In these cells, it was found for the first time that RBV and AgNPs altered the structure of the spherules that contain the viral replication complexes in Golgi stacks. This apparently impaired BUNV replication and as a consequence, diminished the cytopathic effect. At early times post-treatment AgNPs located in the nucleus and in mitochondria and at late times post-treatment they were found in membranous organelles, what could be contributing to the toxicity of the drug. Finally, with plaque assay experiments viruses that were able to replicate in the presence of RBV were isolated. Cells infected with these viruses produced low viral titres and a less severe cytopathic effect than wild type BUNV. The viruses that escaped the RBV treatment do not build their factory in the Golgi complex but in a different organelle. The findings of this investigation demonstrate that the combination of advanced light and electron microscopy methods is of considerable help to unveil the mechanism of action of antiviral drugs and how resistance to antivirals is developed in cells. Detailed studies of toxicity and combined therapies with more than one drug will be necessary to combat emerging and re-emerging viruses.