Final Report: Norwalk Virus-Like Particles (VLPs) for Studying Natural Groundwater Disinfection

EPA Grant Number: R824775
Title: Norwalk Virus-Like Particles (VLPs) for Studying Natural Groundwater Disinfection
Investigators: Grant, Stanley B. , Estes, Mary K. , Olson, Terese M. , Ogunseiten, Oladele
Institution: University of California - Irvine , University of Michigan
EPA Project Officer: Packard, Benjamin H
Project Period: September 1, 1995 through August 1, 1996 (Extended to October 31, 1999)
Project Amount: $230,000
RFA: Water and Watersheds (1995) Recipients Lists
Research Category: Water and Watersheds , Water


An increasing number of communities in the United States rely on recycled wastewater for a portion of their drinking water supply, so-called "potable reuse." Filtration is commonly employed as one of several barriers to the transmission of microbial contaminants in potable reuse systems. The objectives of this study are to: (1) characterize the physicochemical factors that influence the removal of viruses from water by filtration; (2) compare the filtration potential of several different viruses, including indicator viruses (bacteriophage) and virus-like-particle analogs of the important waterborne pathogen Norwalk virus; (3) compare the physicochemical properties and filtration potential of Norwalk virus-like-particles and live Norwalk virus purified from the stools of human volunteers; and (4) develop improved kinetic descriptions of virus filtration suitable for designing filtration systems and predicting the fate and transport of viruses in the subsurface.

Summary/Accomplishments (Outputs/Outcomes):

Physicochemical Factors Influencing Virus Filtration. One of the objectives of this study was to identify the physicochemical variables that determine virus removal in filtration systems. To this end, we conducted laboratory-scale experiments on three different viruses, including two different viruses that infect bacteria, or "bacteriophage," and recombinant Norwalk virus particles generated using a baculovirus expression system. In all cases, we found that pore water chemistry dramatically influenced the filtration of these viruses in packed beds of quartz sand. When the filtration results were combined with microelectrophoresis measurements of the viruses' surface electrical potential, the results were generally consistent with the predictions of classical DLVO theory (i.e., electrostatic interactions between the viruses and filter media ultimately determine a filter's ability to remove viruses from water). From a practical perspective, our results suggest that it may be possible to develop geological and geochemical criteria for the optimal siting of groundwater recharge basins that utilize reclaimed sewage. Furthermore, unit treatment systems could be designed to maximize virus removal by exploiting the intrinsically charged nature of virus particles. These results are described in two journal articles (see Publications/Presentations section), and summarized in more detail below.

Filtration Potential of Different Viruses. Another objective of our study was to characterize how differences in the physical and surface-chemical nature of virus particles affect their removal from water by filtration. Three different virus particles were studied, including Norwalk virus-like-particles, the male-specific coliphage MS2, and a male-specific filamentous coliphage that was isolated from chlorinated effluent of the San Jose Creek Water Reclamation Plant located in Los Angeles County, California. Coliphage are a class of viruses that infect coliform bacteria, and the male-specific coliphage are candidate indicators for viruses that cause human disease. Norwalk virus, on the other hand, is a major cause of waterborne gastrointestinal disease. Virtually nothing is known about the ability of filters to remove Norwalk virus from water because this virus cannot be cultured in the laboratory at the present time. To overcome this methodological obstacle, we utilized Norwalk virus-like-particles (VLPs) that are generated using a biochemical technique in which the expression of the Norwalk virus structural protein leads to the formation of protein particles that resemble live Norwalk virus. These recombinant particles differ from live Norwalk virus in only one known but important respect: they lack genetic material and therefore, cannot initiate a human infection.

Although MS2 and Norwalk virus are morphologically quite similar (i.e., both are spherically shaped particles approximately 30?40 nm in diameter), we found that their surface electrical properties differ significantly. First, the pH at which the Norwalk particles possess zero net charge (the "isoelectric point") is pH 5, roughly 1.5 pH units higher than the isoelectric point of MS2. Secondly, the Norwalk particles are considerably more negatively charged than MS2 at or above neutral pH. Because the pH of most groundwater supplies is typically greater than 7, the electrostatic properties of MS2 and Norwalk virus will differ substantially when these viruses come into contact with groundwater. To determine whether differences in the electrical properties of MS2 and Norwalk particles affect their filtration in porous media, we conducted a set of laboratory-scale filtration experiments using packed beds of quartz sand. We found that the level of virus filtration obtained in a given experiment depended dramatically on electrostatic interactions between the quartz sand (which is negatively charged) and the viruses. In the case of Norwalk virus, for example, the percentage of virus removed from the water by filtration increased from 17 to 99.92 percent when we adjusted the pore water pH downward from 7 to 5?in effect "turning off" the negative charge on the Norwalk particles. By contrast, the filtration of MS2 was relatively insensitive to changes in pore water pH over this range. The importance of this work is threefold: (1) it demonstrates that recombinant Norwalk virus particles can be successfully used for studies of virus filtration; (2) it documents substantial differences in the physicochemical properties of MS2 and Norwalk virus; (3) it demonstrates that differences in the electrostatic properties of viruses translate directly into differences in filtration; and (4) it illustrates that the size and shape of a virus, per se, are relatively poor predictors of filtration potential.

Additional research was carried out on a filamentous male-specific coliphage (SJC-3) that was isolated from treated sewage. The isolate belongs to a class of male-specific coliphage that are naturally present in sources of sewage and hence, may be an indicator of fecal contamination in groundwater. Furthermore, there is some evidence that this class of bacteriophage are mobilized in the subsurface following rainfall events, although the mechanism responsible for this process is not yet clear. Using the same model filtration system of packed columns of quartz sand described above, we found that the filtration of this isolate was strongly dependent on the concentration and valence of the dominant cation in the pore fluid. In one set of experiments involving columns 19 cm in length, virus retention in the column increased from 0 to 99.999 percent when the electrolyte composition of the pore fluid was changed from 10 mM NaCl to 10 mM CaCl2. With one exception, filtration efficiencies calculated from the column experiments were inversely proportional to the electrophoretic mobility of the virus, implying that electrostatic interactions between the virus and the quartz surface dominate the filtration dynamics of this particular bacteriophage. From a practical perspective, these results indicate that small changes in the hardness and total dissolved solids of pore fluids?as might occur following a rainfall event?can impact both the filtration and mobilization of filamentous bacteriophage in subsurface systems. The filtration results with SJC-3 are qualitatively similar to the results obtained with Norwalk VLPs and MS2 described above. In all cases, electrostatic interactions between the virus and mineral surfaces strongly control the efficiency with which viruses are removed from the pore fluid by physicochemical filtration.

Comparison of Norwalk VLPs and Live Norwalk. We also developed a method to label Norwalk VLPs with a fluorescent dye (cy3) and showed that the properties of these particles are similar to non-labeled particles or radiolabeled VLPs based on structure and binding properties to cultured cells. Such particles could be useful for field trials to directly monitor VLP distribution in the subsurface.

Methods to purify native infectious Norwalk virus from the stools of volunteers challenged with virus also were developed. Stools containing the highest levels of virus particles were identified and virus enriched from stools was visualized by electron microscopy. However, purification levels and the concentration of purified virus obtained were not sufficiently high to permit direct comparisons of the filtration properties of these viruses with the VLPs. These efforts are continuing by addition of a step of affinity purification using a column containing immobilized monoclonal antibodies to Norwalk virus. These experiments will continue with funding from another source, and the filtration properties of native virus with VLPs will be compared once a sufficient amount of purified Norwalk virus is obtained.

Fractal Virus Filtration. The studies described above were conducted using well-defined sources of water (distilled water and reagent grade chemicals). To examine how our results obtained with "clean" systems would extrapolate to systems more representative of field conditions, we conducted a suite of packed bed filtration experiments involving three different viruses (the bacteriophage MS2 and PRD-1 and the rNV particles) and two different sources of water (groundwater and highly treated wastewater). In addition, we developed and tested a technique that allowed us, for the first time, to characterize the spatial distribution of viruses retained within the filter. This methodological breakthrough permitted direct testing of the standard filtration theory that is used to predict virus removal in both above ground treatment systems and fate and transport models. These experiments yielded a rich data set that has far-reaching implications for potable reuse, as itemized below.

  1. We discovered that virus filtration does not follow first-order kinetics as commonly assumed, but is better described by a "fractal model" that recognizes the intrinsic heterogeneity of both the viruses and the collectors in natural systems.
  2. The fractal model of filtration predicts a slow power-law decay in virus concentration with distance, instead of the fast exponential decay predicted by standard theory. The power-law nature of virus filtration is consistent with experimental results obtained with the Norwalk VLPs.
  3. From a practical perspective, these results suggest that the first-order kinetic theory routinely used to design potable reuse systems can vastly over-predict the removal of viruses by filtration. Our results also suggest that there is no single rate constant that can be found to characterize virus filtration. Hence, existing models for virus transport in the subsurface (e.g., VIRALT) may be fundamentally flawed.
  4. For the experiments conducted with treated wastewater, we found that natural organic matter (NOM) present in the wastewater significantly attenuated virus filtration, presumably by forming a steric and/or electrosteric barrier to virus deposition. Virus removal efficiencies appear to be strongly affected by the NOM concentration in the wastewater being recycled.

Journal Articles on this Report : 2 Displayed | Download in RIS Format

Other project views: All 15 publications 2 publications in selected types All 2 journal articles
Type Citation Project Document Sources
Journal Article Redman JA, Grant SB, Olson TM, Hardy ME, Estes MK. Filtration of recombinant Norwalk virus particles and bacteriophage MS2 in quartz sand: importance of electrostatic interactions. Environmental Science and Technology 1997;31:3378-3383. R824770 shared with R824775 (Final)
R824775 (Final)
not available
Journal Article Redman JA, Grant SB, Olson TM, Adkins JM, Jackson JL, Castillo MS, Yanko WA. Physicochemical mechanisms responsible for the filtration and mobilization of a filamentous bacteriophage in quartz sand. Water Research, January 1999;33(1):43-52. R824770 shared with R824775 (Final)
R824775 (1998)
R824775 (Final)
not available

Supplemental Keywords:

water, drinking water, groundwater, human health, viruses, hydrology, biology., RFA, Scientific Discipline, Water, Hydrology, Water & Watershed, Environmental Chemistry, Chemistry, Biology, Drinking Water, Watersheds, alternative disinfection methods, microbial contamination, pathogens, public water systems, groundwater disinfection, contaminant transport, human health effects, microbiological organisms, Norwalk, waterborne disease, viruses, NLVs, natural disinfection, microbial pathogens, Norwalk Virus, gastroenteritis, microbial pollution, microbial risk management, aquatic ecosystems, filtration of sewage effluent, drinking water contaminants, drinking water system

Progress and Final Reports:

Original Abstract
  • 1996
  • 1997
  • 1998 Progress Report
  • 1999