Membrane-Based Concentration and Recovery of Viruses from Complex Water Matrices

摘要

Prevention of waterborne disease outbreaks relies on the efficient detection of pathogens in drinking and recreational water. Development of sample concentration technologies that ensure fast and high recovery of pathogens from aquatic samples is crucial for timely detection. The most effective approaches to sample concentration and virus recovery employ membrane filtration and rely on controlling physicochemical interactions between the virus and the filter. The two main goals of the present work were to understand the reasons for the poor efficiency (typically below 30%) of the current methods in recovering human adenovirus and to propose alternative strategies that facilitate concentration and recovery of this important human virus. The first part of the dissertation is devoted to the study on how common methods of virus propagation (broth-based and agar-based) and purification (polyethylene glycol precipitation, centrifugal diafiltration and CsCl density gradient centrifugation) affect physicochemical properties of virions. Experimental data for bacteriophage MS2 showed that results of virus size, charge, and hydrophobicity measurements depend strongly on the methods and protocols used to grow and purify the virus. The optimal sample preparation protocol was determined to consist of broth-based growth followed by purification via CsCl density gradient centrifugation. This method was then used to measure physicochemical properties of human adenovirus 40 (HAdV40) and employ these values to calculate the energy of virion-virion and virion-membrane interactions. The second part of the dissertation describes an experimental study on the recovery of HAdV40 from three water matrices (spiked deionized water, tap water, and high organic content surface water) by crossflow ultrafiltration. Prior to ultrafiltration, membranes were either blocked by calf serum or coated with a polyelectrolyte multilayer to minimize virus adsorption on the membrane surface. The multilayer was designed to be antiadhesive with respect to HAdV 40 using the virus-membrane interaction energy calculations performed earlier. In the sample concentration tests, HAdV 40 was recovered from ultrapure water, tap water, and surface water with very high post-elution recoveries of ∼99%, ∼91%, and ∼84%, respectively. The obtained recovery data were interpreted in terms of physicochemical interactions of HAdV40 virions with the membrane and how components of the eluent disrupt specific interactions between HAdV40 and the membrane to maximize HAdV40 recovery. Results on HAdV40 concentration indicate that the composition of the eluent is the most important factor for achieving high virus recovery and can be designed to efficiently recover viruses even from highly complex water matrices.