Desenvolvimento e padronização de metodologias visando a prospecção e caracterização de novos vírus associados a microalgas e cianobactérias

Abstract

Large nucleocytoplasmic DNA viruses (phylum Nucleocytoviricota) are viruses with unique characteristics that defy the very definition of a virus. This phylum includes chloroviruses, which are viruses with large particles and complex genomes that infect chlorella-like microalgae. Their viral genome contains several genes that encode tRNAs and proteins, including enzymes involved in energy metabolism and the synthesis of macromolecules, as well as hundreds of coding regions (CDS) never before identified in other organisms. Thus, the isolation and characterization of new chloroviruses has great potential for the discovery and characterization of new proteins. Although these viruses have already been identified in various locations and Brazil is a biodiversity hotspot, they have not been explored here. The same occurs with cyanophages, which are viruses that infect cyanobacteria and are important regulators of their abundance and composition. As the discovery and characterization of these aforementioned viruses is so important, it is also essential to develop, adapt and standardize techniques that enable the isolation and characterization of viruses from algae and cyanobacteria. In this work we focused on viruses that infect cyanobacteria of the genus Microcystis, which are widely distributed throughout the world and cause algal blooms, a common phenomenon characterized by exacerbated cell proliferation. Paradoxically, viruses infecting these cyanobacteria have only been isolated in a few eastern countries. Exploring new environments in search of cyanophages can help to understand this diverse group and new isolates can help to solve the gaps related to the phylogeny and biology of these viruses. The objective of this work was to define, test and standardize methods for isolating and characterizing new viruses associated with cyanobacteria and microalgae. To achieve that, algae and cyanobacteria growth curves, cell maintenance tests in different culture media, standardization of plaque assays, prospecting assays and different titration methods were carried out, as well as characterization of cytopathic effects by optical and electron microscopy. In addition, in sílico genomic and phylogenetic analyses of phages that infect Microcystis were carried out. For two of the several environmental samples tested, it was possible to observe cellular alterations indicative of a cytopathic effect, one in Chlorella vulgaris (including the formation of cell lysis plaques in a plaque assay) and the other in Microcystis aeruginosa. Protocols were established for prospecting, cultivating, subculturing and maintaining the cells, as well as preparing samples for Transmission Electron Microscopy, which provides high-resolution images and can be used for all the cells tested. In sílico analysis revealed that the group of cyanophages that infect Microcystis is paraphyletic and their genomes vary substantially, with no syntenic blocks conserved between all cyanophages, nor COGS shared by all. The results of this work suggest that the use of these modified methods may provide conditions for the isolation of the first brazilian viruses infecting algae of the genus Chlorella and cyanophages.