key: cord-0775350-rh86g4jk
authors: Wigginton, Krista Rule; Kohn, Tamar
title: Virus disinfection mechanisms: the role of virus composition, structure, and function
date: 2011-12-09
journal: Curr Opin Virol
DOI: 10.1016/j.coviro.2011.11.003
sha: db1fc7bac6028c1010de19e4251a50870c9486cf
doc_id: 775350
cord_uid: rh86g4jk

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.

Virus disinfection mechanisms: the role of virus composition, structure, and function Krista Rule Wigginton 1 

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.

Obtaining a mechanistic understanding of virus disinfection is a pressing need in environmental engineering owing to the enduring occurrence of waterborne and food-borne virus outbreaks. Many important enteric viruses remain non-culturable to date (e.g. norovirus, hepatitis A); therefore, their susceptibility to disinfection cannot be experimentally tested. Non-culturable virus disinfection kinetics must be either determined with human charge studies or predicted using surrogate viruses that can be cultured in vitro but that differ in composition, structure, and function. A framework that enables the accurate prediction of virus inactivation behavior based on a detailed understanding of the processes involved would assist in the development of effective disinfection strategies.

Scientists have long sought to provide mechanistic descriptions of virus inactivation during drinking water disinfection [1] . In the 1960-1980s, researchers employed scintillation spectroscopy and electron microscopy techniques to detect modifications in viral genomes and proteins and typically reported one of two conclusions: 1) inactivation is the result of damage to the virus proteins or 2) inactivation is the result of damage to the genome [2] [3] [4] [5] [6] . Although these early studies investigated the molecular mechanisms as much as technologically possible, more recent research has focused less on elucidating mechanisms and more on comparing inactivation kinetics with various virus strains, disinfectants, and water chemistries [7] [8] [9] . This is despite the fact that recent technological advances have provided improved tools for probing molecular mechanisms. Collectively, the proposed virus inactivation mechanisms by common water disinfectants vary widely and are often contradictory. For example, the inactivation of poliovirus by chlorine has been attributed to RNA degradation [2] and to capsid protein modifications [10] . At this time, the fundamental questions of what modifications do or do not cause inactivation remain elusive.

Herein, we discuss how the combined knowledge of virus composition, structure and biological function will further our understanding of virus disinfection at the most basic mechanistic level. A physical/chemical description of inactivation is more feasible today than it was ten years ago thanks to advances in genome sequencing, protein mass spectrometry, and structural virology techniques. We focus the majority of our discussion on the disinfection of waterborne enteric viruses [11] , in general, and poliovirus, in particular. Enteric viruses are the most relevant to water treatment and poliovirus has been the focus of numerous disinfection studies over the past several decades [12] [13] [14] . It should be noted that this discussion could be extrapolated to other settings where disinfection is used to mitigate virus transmission, such as food safety or medical equipment sterilization.

such as chlorine, ozone, or UV irradiation are fairly well characterized (Table 1) . Consequently, established reaction rate constants for amino acid and nucleotide monomers can be summed based on their known abundance within virus particles to provide predictions of the relative reaction rates of genome and protein targets [15] . When this is done for Poliovirus 1 Mahoney, chemical disinfectants such as chlorine and ozone are much more reactive with viral protein material than with genomic material (Figure 1 ). On the contrary, UVC radiation affects the genomic material more than the protein material. Unfortunately, such predictions are inaccurate owing to the influence of protein and genome higherlevel organization on reaction rates [16] [17] [18] . For example, when treated with chlorine dioxide, denatured poliovirus genomes degraded at a different rate than native poliovirus genomes [19 ] . Reactions that take place in the genome and proteins during disinfection can form byproducts that further react with amino acids and nucleotides [20] ; this makes reactivity predictions even more complicated.

Unlike chemical oxidants, UVC radiation will lead to direct photolysis of photolabile virus components regardless of their solvent accessibility. A genome-size based approach to predict the sensitivity of virus strains to UVC has been proposed [21] , although others have reported that genome-size does not always correlate with virus susceptibility to UV disinfection [22, 23] . Similar to the genome-size based approach, pyrimidine doublet prevalence in virus genomes was suggested as a framework to predict UVC susceptibility [24 ] . Indeed, a plot of the number of potential dimerization sequences in a virus genome versus effective UV dose suggested a correlation. The presence of outlier virus strains, however, indicates that alternative pathways play a role in some UV inactivation mechanisms. Taken together, these discrepancies demonstrate that virus component information (i.e. genome and protein sequences) alone will not allow for an accurate prediction of susceptibility. A prior knowledge of capsid and genome structure should therefore aid in interpreting and predicting virus particle reactivity and in identifying the particle's most susceptible regions.

Virus disinfection mechanisms Wigginton and Kohn 85 Table 1 Reported second-order rate constants and photochemical constants for the most reactive amino acid and nucleoside monomers with common disinfectants in aqueous solutions at pH $ 7

Chlorine Predicted relative reaction rates for Poliovirus 1 protein and genome components calculated with rate constants and photochemical constants presented in Table 1 .

Coupling structure and composition information aids in our understanding of virus reactivity X-ray crystal structures have been published for numerous enteric viruses [25,26 ,27] and with these reports have come a windfall of valuable information including the location and orientation of capsid protein residues. Cryo-electron microscopy (cryoEM) has expanded our knowledge of virus structures even further, as it allows virologists to study virus particles that are difficult to crystallize due to either complex capsid shapes, inadequate purification, or intermediate, metastable structures (e.g. virus binding or cell entry processes [28, 29 ] ). In fact, recent advances in cryoEM have lead to viral reconstructions at resolutions comparable to those obtained with X-ray crystallography [30, 31 ] . In addition to ordered capsid protein visualization, cryoEM studies have demonstrated that some viruses have ordered genomes [32] and have described specific interactions between capsid proteins and packaged genomes [33] . It should be noted that resolved near-atomic structures are not yet available for some important enteric viruses such as hepatitis A and human norovirus, although recombinant norovirus-like particles have been reconstructed [34] .

Some caution must be taken when using virus structural data to identify the location and solvent accessibility of functional groups. Virus capsids can be fluid in nature and thus functional groups that are normally protected from oxidants in the solvent can be periodically exposed to the capsid surface [35 ] . In human rhinovirus, for example, certain regions are static and in agreement with the crystal structure, while other regions are more fluid [36] .

Together, composition data and structure data will provide an improved framework to describe the reactivity of virus components with disinfectants. Indeed, a number of studies on nonviral proteins have used structural data to explain the site-specific reactivity of protein components [16, 17, 37, 38] . At this time, however, only a few reports on virus disinfection have mentioned resolved virus structures in the interpretation of results [10,39 ,40 ] . A study on adenovirus inactivation with chlorine did employ adenovirus structural data to suggest that damaged Met or His residues near a critical motif may contribute to inactivation [39 ] . In another study, protein mass spectrometry was used to identify specific residues in MS2's capsid proteins as the virus was inactivated by UV 254 and singlet oxygen [40 ] . Residues on the outside surface of the capsid were modified with 1 O 2 treatment while residues on the inside surface of the capsid near the viral genome were modified with UV treatment. More research is clearly needed to elucidate the effect of virus structure on the reactivity of virus components.

Composition and structure information aids in describing where modifications are most likely to occur in a virus particle during disinfection; however, modifications do not always cause inactivation. For example, although ozonation of poliovirus altered viral proteins VP1 and VP2, inactivation was ultimately attributed to genome damage [5] . Solely identifying susceptible virus regions based on composition and structure will be insufficient to describe and predict the effect of a disinfectant on virus infectivity. One must also consider the effect that particular modifications have on fundamental virus functions (e.g. host-cell binding, genome entry, etc.). In order to do this, the fundamental functions and virus components involved in those functions must be well defined.

Virus structural and dynamic information has provided an improved understanding of the biological function of viral domains, including virus-host cell interaction [26 ,41 ] , virus assembly [26 ] , capsid-RNA interaction sites [33, 42 ] and RNA release ( [42 ] and references therein). This new knowledge provides valuable insight into critical structures and biological functions of the virus and thus identifies regions and functions that should be targeted in virus neutralization or inactivation strategies.

The promise of a structure/function-based approach for disinfection is underscored by the fact that the medical field has exploited this method to develop vaccines [41 ] and antiviral drugs [43] as well as to better understand the mechanism of previously developed vaccines [41 ] . This type of approach has great benefits over the traditional method of screening and isolating fortuitously emerged, non-virulent virus strains or neutralizing antibodies. Namely, it enables the rational design of site-specific antivirals and vaccines that target relevant virus structures common to several strains or species. For example, coronavirus strains were long believed to have host receptors that were too diverse and too prone to mutation to be susceptible to a broad-spectrum antiviral. Recently, however, Yang et al. [44 ] used function and structure information of all three genetic coronavirus clusters to determine that the main protease has a highly conserved substrate-recognition pocket. Based on this structural information combined with compositional information of conserved amino acids within this region, a protease inhibitor was designed that successfully prevented virus replication of two coronaviruses. Ultimately, the authors suggest that the knowledge of the conserved structure and biochemistry of the protease will lead to the development of a single, broad spectrum antiviral that targets all coronaviruses. A similar approach to water disinfection will lead to the rational design of novel disinfectants.

An important difference between water disinfection and antiviral drugs, however, is that traditional disinfection does not operate by physically blocking a virus site or particular function via the addition of an external chemical or antibody, but by chemically or structurally altering one or several important sites. We therefore expect that the intrinsic reactivities of the various virus components are of greater importance to disinfection than to drug development. As discussed above, this intrinsic reactivity is dictated by both specific chemical composition and the structure. As such, the structure/function approach used in the medical field would have to be expanded to a composition/structure/function approach in water treatment.

As an example of how structural commonalities and compositional differences may be used to predict susceptibilities to disinfectants, we look at poliovirus 1 Brunhilde and poliovirus 1 Mahoney. The Brunhilde strain was reported to be twice as resistant to chlorination as the Mahoney strain [45, 46] . This discrepancy in inactivation kinetics is intriguing as the two strains have nearly identical capsid protein sequences ($98%) and have similarly sized genomes. Inactivation of the Mahoney strain by chlorine was attributed to a loss in ability to attach to the host cell [10] , however this has not been examined for the Brunhilde strain. CryoEM analysis has provided a three-dimensional structure of the poliovirus particle bound to its host cell receptor, sPvr [47 ] , and a number of capsid protein residues were implicated in the virus capsid-host cell interaction. Interestingly, six of these implicated residues differ in the Mahoney and Brunhilde strains, including Mahoney VP2 Met140 (Thr in Brunhilde) and His141 (Tyr in Brunhilde) ( Figure 2 ). Methionine and histidine side chains readily react with chlorine; threonine and tyrosine sidechains, on the contrary, are much less reactive. It is thus possible that the lack of Met and His residues at the binding site is responsible for the greater resistance of the Brunhilde strain to chlorine disinfection. This is only a hypothetical explanation and will need to be confirmed through experimentation; it does, however, demonstrate the manner in which composition, structure, and function information can assist in deducing virus inactivation mechanisms. It also demonstrates how such knowledge aids in predicting how a new strain will behave based on mutations in the binding site. Suppose, for example, a new poliovirus strain emerges with fewer reactive functional groups in its binding site. The new strain would be expected to be more resistant to disinfection than the well-characterized strain. For fast-mutating viruses like human norovirus, this type of predictive tool would be particularly valuable. Research is needed to assess exactly how similar virus strains should be in structure, composition, and function for such a predictive tool to work.

To reach a point where a holistic understanding of virus inactivation is in hand, a number of questions will first need to be addressed. Specific questions include: 1) Which virus protein residues are involved with fundamental functions and how do these vary amongst different strains and species; 2) What specific chemical modifications take place in the genome and capsid during disinfection and what effects do these modifications have on virus structure and function; 3) How similar are disinfectant-induced modifications amongst various enteric viruses? Answering these questions is a lofty goal given the numerous virus-disinfectant pairs that will need to be examined.

In conclusion, the study of virus fate in water treatment is entering an exciting new phase thanks to the advent of tools that provide insight into virus structure and function. As a result, virus inactivation predictive tools and the design of highly efficient disinfectants may be a reality in the not-so-distant future.

Papers of particular interest, published within the period of review, have been highlighted as:

of special interest of outstanding interest Virus disinfection mechanisms Wigginton and Kohn 87

Current Opinion in Virology

Poliovirus Mahoney strain capsid. VP2 Met140 is highlighted in green and VP2 His141 is highlighted in red. Structural visualization with PYMOL software (http:/www.pymol.org).

Molecular mechanisms of viral inactivation by water disinfectants

Structural and compositional changes associated with chlorine inactivation of polioviruses

Mechanism of disinfectionincorporation of Cl-36 into F2 virus

Mechanism of ozone inactivation of bacteriophage-F2

Mechanism of enteroviral inactivation by ozone

Mechanisms of inactivation of poliovirus by chlorine dioxide and iodine

Effects of source water quality on chlorine inactivation of adenovirus, coxsackievirus, echovirus, and murine norovirus

Inactivation of adenoviruses, enteroviruses, and murine norovirus in water by free chlorine and monochloramine

Inactivation of enteric adenovirus and feline calicivirus by chlorine dioxide

Capsid functions of inactivated human picornaviruses and feline calicivirus

Enterically infecting viruses: pathogenicity, transmission and significance for food and waterborne infection

Inactivation by bromine of single poliovirus particles in water

Relative resistance to chlorine of poliovirus and coxsackievirus isolates from environmental sources and drinking water

Reduction of Norwalk virus, poliovirus 1, and bacteriophage MS2 by ozone disinfection of water

Singlet oxygen-mediated damage to proteins and its consequences

Oxidative modification of cytochrome c by singlet oxygen

Reactivity of singlet oxygen toward proteins: the effect of structure in basic pancreatic trypsin inhibitor and in ribonuclease A

Photoinduced DNA cleavage via electron transfer: demonstration that guanine residues located 5' to guanine are the most electron-donating sites

Degradation of the Poliovirus 1 genome by chlorine dioxide

This study compared the susceptibility of extracted viral genomes with viral genomes inside an intact virus when treated with chlorine dioxide. The results offered evidence that the structure of the viral genome plays a role in the genome's susceptibility to oxidant attack

Hypochlorous acidmediated protein oxidation: how important are chloramine transfer reactions and protein tertiary structure?

Predicted inactivation of viruses of relevance to biodefense by solar radiation

Inactivation of bacteriophages in water by means of non-ionizing (UV-253.7 nm) and ionizing (gamma) radiation: a comparative approach

Inactivation of poliovirus 1 and F-specific RNA phages and degradation of their genomes by UV irradiation at 254 nanometers

A genomic model for predicting the ultraviolet susceptibility of viruses

This study proposed a mathematical model to predict the susceptibility of viruses to inactivation by UV 254 . The model was based on the prevalence of viral genome sequences that readily undergo dimerization with UV radiation

The crystal structure of coxsackievirus A9: new insights into the uncoating mechanisms of enteroviruses. Structure

Biological and immunological characteristics of hepatitis E virus-like particles based on the crystal structure

This research solved the first crystal structure of hepatitis E virus-like particles at 3.5-Å resolution and identified a putative cellular binding region in the capsid via mutational analyses

Crystal structure of human adenovirus at 3.5 Å resolution

3.3 Å cryo-EM structure of a nonenveloped virus reveals a priming mechanism for cell entry

Viral life cycles captured in threedimensions with electron microscopy tomography

An effective review of recent electron cryotomography studies that have elucidated the complex structure and life cycles of viruses

Near-atomic resolution using electron cryomicroscopy and single-particle reconstruction

Near-atomic resolution cryo-EM for molecular virology

A review highlighting several recent cryo-EM virus studies and the novel insights they have provided

Visualization of ordered genomic RNA and localization of transcriptional complexes in rotavirus

Atomic structure reveals the unique capsid organization of a dsRNA virus

X-ray crystallographic structure of the Norwalk virus capsid

Viral capsid mobility: a dynamic conduit for inactivation

This study provided evidence that virus inactivation by small alkylating agents is due to reactions in the viral genome; it therefore demonstrated that certain chemicals can diffuse through virus protein capsids even when the capsids are void of holes or major crevices

Capsid structure and dynamics of a human rhinovirus probed by hydrogen exchange mass spectrometry

Hypochlorous acid oxidizes methionine and tryptophan residues in myoglobin

Site-specific hypochlorous acid-induced oxidation of recombinant human myoglobin affects specific amino acid residues and the rate of cytochrome b5-mediated heme reduction

Mechanistic aspects of adenovirus serotype 2 inactivation with free chlorine

This study examined the mechanisms responsible for Adenovirus type 2 inactivation with free chlorine disinfection. Inactivation was attributed to damage in virus proteins that control functions post host cell attachment

Oxidation of virus proteins during UV 254 and singlet oxygen mediated inactivation

This study used protein mass spectrometry to detect modifications in virus capsid proteins as virus were inactivated and demonstrated that singlet oxygen and UVC affect different virus components

Crystal structure of measles virus hemagglutinin provides insight into effective vaccines

This study solved the crystal structure of measles virus hemagglutinin-the attachment protein responsible for virus entry into the host cell and a target for neutralizing antibodies. The authors suggested that the solved molecular structure can aid in structure-based measles vaccine development

Catching a virus in the act of RNA release: a novel poliovirus uncoating intermediate characterized by cryo-electron microscopy

Emerging antiviral targets for influenza A virus

Design of wide-spectrum inhibitors targeting coronavirus main proteases

Irreversible inhibitors for coronavirus strains were designed using the structure of a conserved substrate-recognition pocket. The results demonstrate that it is possible to develop a single antiviral agent effective against a number of coronavirus strains

Inactivation of poliovirus-I (Brunhilde) single particles by chlorine in water

Inactivation by chlorine of single poliovirus particles in water

Threedimensional structure of poliovirus receptor bound to poliovirus

The structure of poliovirus type 1 bound to its cellular receptor, sPvr, was determined at 21-Å resolution with cryo-EM. Capsid protein residues involved in the poliovirus-sPvr interactions were identified and were in general agreement with previous mutagenesis results

Hypochlorous acid interactions with thiols, nucleotides, DNA, and other biological substrates

Degradation of nucleic-acids with ozone. V. Mechanism of action of ozone on deoxyribonucleoside 5'-monophosphates

Rate constants of ozone reactions with DNA, its constituents and related compounds

PhotochemCADz: a computer-aided design and research tool in photochemistry

DNA excited-state dynamics: from single bases to the double helix

Gorner H: Photochemistry of DNA and related biomoleculesquantum yields and consequences of photoionization

Interactions of hypochlorous acid with pyrimidine nucleotides, and secondary reactions of chlorinated pyrimidines with GSH, NADH, and other substrates

Absolute rate constants for the reaction of hypochlorous acid with protein side chains and peptide bonds

Kinetics of ozonation. 2. Amino-acids and model compounds in water and comparisons to rates in nonpolar-solvents

Numerical values of absorbances of aromatic amino acids in acid neutral and alkaline solutions

UV photolysis of aromatic amino acids and related dipeptides and tripeptides