Charge, size distribution and hydrophobicity of viruses: Effect of propagation and purification methods

Abstract

Two virus propagation methods (in broth and on double agar overlay) and three purification procedures (PEG precipitation, centrifugal diafiltration and CsCl density gradient centrifugation) were comparatively evaluated using MS2 and P22 bacteriophages as model viruses. The prepared stocks were characterized in terms of electrophoretic mobility as a function of pH, particle size distribution, surface tension components and the overall hydrophobicity of the virus, as well as the percentage of infectious and total virus recovered. The obtained data were used to rank the purification methods according to six criteria of likely practical relevance. Regardless of the purification method applied, virus propagation in broth media resulted in higher purity virus stocks as the growth on double agar overlay introduced difficult-to-remove residual agar. CsCl density gradient centrifugation gave the highest quality bacteriophage suspensions, recovered infectious P22 at least as efficiently as the other two purification methods and selected for intact P22 virions over damaged ones. The impurities remaining in the virus suspension after PEG precipitation and centrifugal diafiltration broadened the size distribution and interfered with electrophoretic mobility measurements. The residual impurities had a major impact on the free energy of virus-virus interfacial interaction (the quantitative measure of virus hydrophobicity/hydrophilicity) leading to an incorrect determination of P22 bacteriophage as hydrophilic. The trends in measured physicochemical properties can be rationalized by considering impurity-coated virions as permeable soft particles.