Krista Wigginton

Nanomaterial Enabled Biosensors for Pathogen Monitoring - A Review

One promising, but currently underexplored, area for the future of drinking water pathogen monitoring stems from the development of nanomaterial-enabled detection strategies. The nanoscience literature contains numerous reports of nanoenabled biosensors; however, to date only a small percentage have focused on the detection of whole cells, in general, and waterborne pathogens, in particular. There are significant opportunities for the use of nanoenabled biosensors for environmental monitoring, and this review is intended to both illustrate the state of this field and to spur additional research in this area.

Virus inactivation mechanisms: impact of disinfectants on virus function and structural integrity

Oxidative processes are often harnessed as tools for pathogen disinfection. Although the pathways responsible for bacterial inactivation with various biocides are fairly well understood, virus inactivation mechanisms are often contradictory or equivocal. In this study, we provide a quantitative analysis of the total damage incurred by a model virus (bacteriophage MS2) upon inactivation induced by five common virucidal agents (heat, UV, hypochlorous acid, singlet oxygen, and chlorine dioxide). Each treatment targets one or more virus functions to achieve inactivation: UV, singlet oxygen, and hypochlorous acid treatments generally render the genome nonreplicable, whereas chlorine dioxide and heat inhibit host-cell recognition/binding. Using a combination of quantitative analytical tools, we identified unique patterns of molecular level modifications in the virus proteins or genome that lead to the inhibition of these functions and eventually inactivation. UV and chlorine treatments, for example, cause site-specific capsid protein backbone cleavage that inhibits viral genome injection into the host cell. Combined, these results will aid in developing better methods for combating waterborne and foodborne viral pathogens and further our understanding of the adaptive changes viruses undergo in response to natural and anthropogenic stressors.

Oxidation of virus proteins during UV254 and singlet oxygen mediated inactivation.

Despite the widespread use of UV(254) irradiation and solar disinfection for water treatment, little is known about the photochemical pathways that lead to virus inactivation by these treatments. The goal of this study was to identify reactions that occur in virus capsid proteins upon treatment by UV(254) irradiation and (1)O(2), an important oxidant involved in sunlight-mediated disinfection. Bacteriophage MS2 was inactivated via UV(254) irradiation and exposure to (1)O(2) in buffered water, and their capsid proteins were then analyzed with MALDI-TOF-TOF and ESI-TOF before and after digestion with protease enzymes. The results demonstrate that chemical modifications occur in the MS2 major capsid protein with both treatments. One oxidation event was detected following (1)O(2) treatment in an amino acid residue located on the capsid outer surface. UV(254) treatment caused three chemical reactions in the capsid proteins, two of which were oxidation reactions with residues on the capsid outer surface. A site-specific cleavage also occurred with UV(254) irradiation at a protein chain location on the inside face of the capsid shell. We attribute this UV(254) induced protein scission, which is nearly unprecedented in the literature, to a close association between the affected residues and viral RNA, an efficient UV(254) absorber. These results suggest that viral protein oxidation by UV(254) and (1)O(2) may play a role in virus inactivation and that viral inactivation may be tracked with mass spectrometric measurements.

Virus disinfection mechanisms: the role of virus composition, structure, and function

Drinking waters are treated for enteric virus via a number of disinfection techniques including chemical oxidants, irradiation, and heat, however the inactivation mechanisms during disinfection remain elusive. Owing to the fact that a number of significant waterborne virus strains are not readily culturable in vitro at this time (e.g. norovirus, hepatitis A), the susceptibility of these viruses to disinfection is largely unknown. An in-depth understanding of the mechanisms involved in virus inactivation would aid in predicting the susceptibility of non-culturable virus strains to disinfection and would foster the development of improved disinfection methods. Recent technological advances in virology research have provided a wealth of information on enteric virus compositions, structures, and biological functions. This knowledge will allow for physical/chemical descriptions of virus inactivation and thus further our understanding of virus disinfection to the most basic mechanistic level.

Impact of virus aggregation on inactivation by peracetic acid and implications for other disinfectants

Viruses in wastewater and natural environments are often present as aggregates. The disinfectant dose required for their inactivation, however, is typically determined with dispersed viruses. This study investigates how aggregation affects virus inactivation by chemical disinfectants. Bacteriophage MS2 was aggregated by lowering the solution pH, and aggregates were inactivated by peracetic acid (PAA). Aggregates were redispersed before enumeration to obtain the residual number of individual infectious viruses. In contrast to enumerating whole aggregates, this approach allowed an assessment of disinfection efficiency which remains applicable even if the aggregates disperse in post-treatment environments. Inactivation kinetics were determined as a function of aggregate size (dispersed, 0.55 and 0.90 μm radius) and PAA concentration (5-103 mg/L). Aggregation reduced the apparent inactivation rate constants 2-6 fold. The larger the aggregate and the higher the PAA concentration, the more pronounced the inhibitory effect of aggregation on disinfection. A reaction-diffusion based model was developed to interpret the experimental results, and to predict inactivation rates for additional aggregate sizes and disinfectants. The model showed that the inhibitory effect of aggregation arises from consumption of the disinfectant within the aggregate, but that diffusion of the disinfectant into the aggregates is not a rate-limiting factor. Aggregation therefore has a large inhibitory effect if highly reactive disinfectants are used, whereas inactivation by mild disinfectants is less affected. Our results suggest that mild disinfectants should be used for the treatment of water containing viral aggregates.

Subtle differences in virus composition affect disinfection kinetics and mechanisms

ABSTRACT Viral disinfection kinetics have been studied in depth, but the molecular-level inactivation mechanisms are not understood. Consequently, it is difficult to predict the disinfection behavior of nonculturable viruses, even when related, culturable viruses are available. The objective of this work was to determine how small differences in the composition of the viral genome and proteins impact disinfection. To this end, we investigated the inactivation of three related bacteriophages (MS2, fr, and GA) by UV254, singlet oxygen (1O2), free chlorine (FC), and chlorine dioxide (ClO2). Genome damage was quantified by PCR, and protein damage was assessed by quantitative matrix-assisted laser desorption ionization (MALDI) mass spectrometry. ClO2 caused great variability in the inactivation kinetics between viruses and was the only treatment that did not induce genome damage. The inactivation kinetics were similar for all viruses when treated with disinfectants possessing a genome-damaging component (FC, 1O2, and UV254). On the protein level, UV254 subtly damaged MS2 and fr capsid proteins, whereas GAs capsid remained intact. 1O2 oxidized a methionine residue in MS2 but did not affect the other two viruses. In contrast, FC and ClO2 rapidly degraded the capsid proteins of all three viruses. Protein composition alone could not explain the observed degradation trends; instead, molecular dynamics simulations indicated that degradation is dictated by the solvent-accessible surface area of individual amino acids. Finally, despite the similarities of the three viruses investigated, their mode of inactivation by a single disinfectant varied. This explains why closely related viruses can exhibit drastically different inactivation kinetics.

Gold-coated polycarbonate membrane filter for pathogen concentration and SERS-based detection

A SERS-based method for the concentration and detection of Giardia lamblia cysts in finished drinking water is reported. In this method, samples are concentrated with a membrane filter and then cysts captured on the filter surface are labeled with immunogold SERS labels and quantified via Raman spectroscopy. Anodisc((R)) membrane filters, silver membrane filters, and electroless gold-coated polycarbonate track etched (PCTE) membrane filters were investigated for their compatibility with the SERS based detection strategy. The largest pore size Anodisc((R)) membrane commercially available was too small for the proposed method because they led to physical retention of immunogold. When silver membrane filters were employed, cysts were difficult to distinguish from nonspecifically bound labels and cyst recovery from distilled water samples was only approximately 12.3%. With gold-coated PCTE membranes, however, cysts were readily detectable and cyst recovery was approximately 95%. This Raman based method simplifies Giardia detection and has potential to be extended to the simultaneous detection of numerous pathogenic organisms. To our knowledge, this is the first report coupling the use of membrane filters for the concentration and detection of organisms from water samples with a SERS based detection strategy.

Halogenation of bisphenol-A, triclosan, and phenols in chlorinated waters containing iodide

Free chlorine reacts with the iodide (I(-)) present in disinfected waters to produce free iodine. Past research has indicated that this nonchlorinated oxidant exhibits greater reactivity and potentially produces more toxic byproducts than free chlorine alone. In this study, we examined the reactivity of the phenolic compounds 2,4-dichlorophenol, triclosan, and bisphenol A in chlorinated waters containing I(-). The free iodine mediated reactions were probed as a function of the initial I(-) concentration and the solution pH. Over the pH range of 5.5 to 10 for an initial I(-) concentration of 10 μM, the observed transformation kinetics of 2,4-dichlorophenol were generally 2-15× faster than reactions with free chlorine alone, while for triclosan and bisphenol A the free iodine mediated transformations were ≈3-20× and ≈230-660× faster, respectively. A comprehensive reaction model that simultaneously accounts for free chlorine and free iodine reactivity in these systems was developed to determine second-order rate constants for the chlorinated and iodinated oxidants. For all test compounds, iodinated daughter products are rapidly produced when free chlorine reacts in the presence of I(-).

UV Radiation Induces Genome-Mediated, Site-Specific Cleavage in Viral Proteins

Much research has been dedicated to understanding the molecular basis of UV damage to biomolecules, yet many questions remain regarding the specific pathways involved. Here we describe a genome‐mediated mechanism that causes site‐specific virus protein cleavage upon UV irradiation. Bacteriophage MS2 was disinfected with 254 nm UV, and protein damage was characterized with ESI‐ and MALDI‐based FT‐ICR, Orbitrap, and TOF mass spectroscopy. Top‐down mass spectrometry of the products identified the backbone cleavage site as Cys46–Ser47 in the virus capsid protein, a location of viral genome–protein interaction. The presence of viral RNA was essential to inducing backbone cleavage. The similar bacteriophage GA did not exhibit site‐specific protein cleavage. Based on the major protein fragments identified by accurate mass analysis, a cleavage mechanism is proposed by radical formation. The mechanism involves initial oxidation of the Cys46 side chain followed by hydrogen atom abstraction from Ser47 Cα. Computational protein QM/MM studies confirmed the initial steps of the radical mechanism. Collectively, this study describes a rare incidence of genome‐induced protein cleavage without the addition of sensitizers.

Survivability, Partitioning, and Recovery of Enveloped Viruses in Untreated Municipal Wastewater

Many of the devastating pandemics and outbreaks of the 20th and 21st centuries have involved enveloped viruses, including influenza, HIV, SARS, MERS, and Ebola. However, little is known about the presence and fate of enveloped viruses in municipal wastewater. Here, we compared the survival and partitioning behavior of two model enveloped viruses (MHV and ϕ6) and two nonenveloped bacteriophages (MS2 and T3) in raw wastewater samples. We showed that MHV and ϕ6 remained infective on the time scale of days. Up to 26% of the two enveloped viruses adsorbed to the solid fraction of wastewater compared to 6% of the two nonenveloped viruses. Based on this partitioning behavior, we assessed and optimized methods for recovering enveloped viruses from wastewater. Our optimized ultrafiltration method resulted in mean recoveries (±SD) of 25.1% (±3.6%) and 18.2% (±9.5%) for the enveloped MHV and ϕ6, respectively, and mean recoveries of 55.6% (±16.7%) and 85.5% (±24.5%) for the nonenveloped MS2 and T3, respectively. A maximum of 3.7% of MHV and 2% of MS2 could be recovered from the solids. These results shed light on the environmental fate of an important group of viruses and the presented methods will enable future research on enveloped viruses in water environments.