Physical fractionation of aquatic viral assemblages

Abstract

Aquatic viruses are extraordinarily diverse and have major influences on plankton ecology and evolution, but the majority of these viruses remain uncharacterized. Most viruses have not been cultivated and cultivationindependent means to explore viral diversity have provided only a fragmented view of viral genomic information. In this study, ultracentrifugation and chromatographic methods were evaluated for their ability to fractionate aquatic viruses based on their differing physical properties with the aim of physically enriching subsets of aquatic viruses for further study. Centrifugation in continuous cesium chloride gradients, strong and weak anion‐exchange chromatography, and size‐exclusion chromatography with Sephacryl S‐1000 were found to separate aquatic viruses based on their differing buoyant densities, surface charges, and sizes, respectively. Sucrose density gradients, cation‐exchange chromatography, and size‐exclusion chromatography using controlled‐pore glass were found to have insufficient resolution to effectively separate viral assemblages. Using the successful methods listed above, we were able to separate complex assemblages of viruses into fractions that had distinguishable subsets of the viruses present in the original community as determined by pulsed‐field gel electrophoresis. Strong anion‐exchange chromatography was particularly effective at separating viruses in the samples and resulted in one to six dominant viral genome bands in most fractions. Our results suggest that individual populations of viruses may be physically enriched, and possibly isolated, from complex assemblages without cultivation. These fractionation methods are expected to improve our ability to characterize the genotypes and phenotypes of the uncultivated majority of viruses in aquatic environments.

Assembly of a Marine Viral Metagenome after Physical Fractionation

“…In the initial continuous cesium chloride (CsCl) gradient of the viral concentrate, a large portion of the viruses banded with little resolution over a broad range and at high densities (1.47–1.56 g ml −1 ; data not shown), an atypical result for this method [23]. The presence of a viscous whitish matter in this region of the gradient suggested that the viruses could be adsorbed to an unknown substance.…”

“…Continuous cesium chloride (CsCl) gradients were used as the first fractionation step to separate viruses from one another based on their differing buoyant densities [23]. The density of the viral concentrate was adjusted to 1.45 g ml −1 by the addition and dissolution of solid molecular grade CsCl (Fisher Scientific) and 10.5 ml of the resulting solution was deposited into a 12-ml polyallomer ultracentrifuge tube (Beckman Coulter).…”

“…A fraction from the continuous CsCl gradient was selected for further separation of viruses by strong anion-exchange chromatography [23]. A BioLogic HR Workstation (Bio-Rad) equipped with a 1-ml sample injector, gradient mixer, fraction collector, and UV and conductivity meters was used to run a step gradient through an UNO Q1 strong anion-exchange chromatography column (Bio-Rad).…”

“…Multi-dimensional physical fractionation of natural aquatic viral assemblages can be achieved by exploiting differences in the sizes, surface charges, and buoyant densities among different populations of viruses [23]. Here, we use two physical fractionation steps in series to enrich a limited number of viral consortia from a complex marine assemblage in order to test whether such a procedure would result in a high proportion of assembled sequences.…”

Global morphological analysis of marine viruses shows minimal regional variation and dominance of non-tailed viruses

“…Several existing and emerging approaches will likely help identify and characterize non-tailed marine viruses. These include culture-based approaches (for example, targeted isolations with existing and new marine bacterial, archaeal and eukaryotic cultures), as well as new methods that either require only the host to be in culture (for example, viral tagging; Deng et al, 2012) or are completely cultivation-independent (for example, physical fractionation of viral assemblages; Bergeron et al, 2007;Steward and Rappé, 2007;Brum and Steward, 2011;Brum et al, 2013). The abundance and distribution of genetically characterized, non-tailed viruses could also be explored using phageFISH (Allers et al, 2013).…”