Viral concentration methods for diversity studies in soil samples

Abstract Soil is the greatest reservoir of microorganisms, which are the main agents responsible for biogeochemical cycles, as well as for the decomposition of organic compounds and the supply of plant nutrients. Among all the microorganisms present in soil are the viruses, which influence the activity and ecology of other populations in the environment. As bacteria are the most common host of viruses in soil, most virus-like particles (VLPs) are considered to be bacteriophages. Since they can have an effect on the activity and ecology of bacteria, they can influence bacterial evolution by transduction, a mechanism of horizontal gene transfer in which the bacteriophage transfers DNA from one bacteria to another. Although viruses are the most abundant among all microorganisms and have great importance in the environment, viral diversity has not been well explored in soil. Because of this fact, the aim of the present study was to determine the best concentration method for the study of viral diversity. For that purpose, three concentration methods commonly used for viral study were selected and adapted to viral concentrations in soil samples: negatively charged HA membranes, polyethylene glycol (PEG) and ultracentrifugation (UF) were used, and viral diversity was evaluated by the random amplified polymorphic DNA (RAPD), transmission electron microscopy (TEM) and metagenomic approaches. Of the three concentration methods, the HA membrane showed the best results, more diverse in metagenomic analysis and with more VLP morphotypes, followed by ultracentrifugation. These techniques recovered viral particles of over 100 nm in size, while PEG recovered smaller particles. RAPD and TEM analyses were shown to be effective methods for the study of viral diversity, corroborating the data obtained by sequencing.