Critical Review pubs.acs.org/est
Viral Aggregation: Impact on Virus Behavior in the Environment Charles P. Gerba* and Walter Q. Betancourt* Department of Soil, Water and Environmental Science Water & Energy Sustainable Technology (WEST) Center, The University of Arizona, 2959 W. Calle Agua, Nueva Tucson, Arizona 85745, United States ABSTRACT: Aggregates of viruses can have a significant impact on quantification and behavior of viruses in the environment. Viral aggregates may be formed in numerous ways. Viruses may form crystal like structures and aggregates in the host cell during replication or may form due to changes in environmental conditions after virus particles are released from the host cells. Aggregates tend to form near the isoelectric point of the virus, under the influence of certain salts and salt concentrations in solution, cationic polymers, and suspended organic matter. The given conditions under which aggregates form in the environment are highly dependent on the type of virus, type of salts in solution (cation, anion. monovalent, divalent) and pH. However, virus type greatly influences the conditions when aggregation/disaggregation will occur, making predictions difficult under any given set of water quality conditions. Most studies have shown that viral aggregates increase the survival of viruses in the environment and resistance to disinfectants, especially with more reactive disinfectants. The presence of viral aggregates may also result in overestimation of removal by filtration processes. Virus aggregation-disaggregation is a complex process and predicting the behavior of any individual virus is difficult under a given set of environmental circumstances without actual experimental data.
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INTRODUCTION The existence of viral aggregates or aggregates has been suggested more than 50 years ago, and has been used to explain various phenomena on virus behavior such as failure of antibodies to neutralize viruses in suspension and resistance of subpopulations to disinfectants. Probably the most noted case of failure to consider about the importance of viral aggregates was the acquisition of paralytic poliomyelitis in children from formalin treated Sabin vaccine in the 1950s in the United States.1,2 Viral aggregates have been used to explain the nonlinear nature of virus inactivation in the environment and resistance to disinfectants.3−5 While other explanations6 have been suggested for this effect, aggregates still seem to be the most common explanation. Understanding this phenomenon is important in assessing the needed doses of disinfectants to inactivate infectious viruses, assess fate and transport in the environment and removal by water and wastewater treatment processes.
specimens of persons infected with enteric viruses (adenovirus, rotavirus, astrovirus) 80% of the viruses were in aggregates of 10−15 virions, and 45% were in aggregates of greater than 50 virions.10 Aggregates of viruses enveloped by cell debris have also been observed.12 It is likely that viruses produced in this state are released into the environment, thereby remaining stable after excretion.8 The type of cell line in which a particular virus is grown may also affect the occurrence of aggregates.13 Even the strain of a virus can exhibit different degrees of aggregation depending upon the addition of a salt solution with specific concentration and type of cation or anion, that is, monovalent, divalent.14−17 Individual virions in suspension may be induced to aggregate by changes in pH, salt concentration, type and concentration of cations, natural organic matter, and polyelectrolytes (Table 1; Figure 1). Both electrostatic and hydrophobic forces are believed to play a role in aggregate formation. Viruses in suspension tend to aggregate near or at their isoelectric point where charge on the virus is near neutral and where repulsive electrostatic forces are the least. The isoelectric point of viruses varies from 1.9 to 8.4,18 thus pH of the water can affect the potential for aggregate formation differently for different viruses. The types of salts and salt concentrations will also affect the charge on the virus by reducing repulsive electrostatic charges of the proteins on the virus surface.19 Monovalent cations have less of an effect on aggregation.20 A stronger effect in aggregate formation for MS2 coliphage,21 rotavirus10
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TYPES AND FORMATION OF VIRAL AGGREGATES Viral aggregates may originate in several different ways. Electron micrographs of infected cells have shown large viral aggregates in the interior of the cells.7 Such aggregates may consist of hundreds of virions. Picornaviruses in particular are produced intracellularly in tightly packed formations.8,9 Aggregates are likely released when the cells rupture. Such aggregates when released into the intestine may also become associated with particulate organic matter. Large aggregates of up to 1000 nm in radius of adenovirus whose viral particles have an overall diameter of 70−90 nm or rotavirus (60−80 nm in diameter) and astrovirus (28−30 nm in diameter) have been observed in fecal specimens.10,11 In an examination of stool © 2017 American Chemical Society
Received: Revised: Accepted: Published: 7318
December 24, 2016 June 1, 2017 June 9, 2017 June 9, 2017 DOI: 10.1021/acs.est.6b05835 Environ. Sci. Technol. 2017, 51, 7318−7325
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Environmental Science & Technology
structure of the virus can also effect aggregate formation. The presence of fibers (“pentons”) of the surface of adenovirus was suggested to increase the energy barrier because of steric forces created by the fibers for electrostatic repulsion resulting in less aggregation.20,31−33 Moreover, the presence of spikes on coliphage phiX-174 was also suggested to result in less compact aggregates causing them to be less resistant to disinfection than aggregates of MS2 coliphage.34,35 Formation of aggregates may also be influenced by temperature. Some studies report greater formation of aggregates at higher temperatures because of greater Brownian movement,33 although poliovirus was found to aggregate upon prolonged storage at 4 °C and repeated freezing and thawing.36 Other factors such as virion size, virus type, presence of solutes, particulates, and concentration of the viral particles may influence aggregate formation of viruses at any given temperature.37 Disaggregation can also occur by changes in these physical-chemical factors, but some appear to be stable even with changing water quality conditions.33,38,39 Aggregates may form among viruses of the same type, different strains of the same virus and different species. Poliovirus aggregates were not dispersed by addition to lake or distilled water, but did disperse when added to phosphate buffer or secondarily treated sewage effluent.8 Floyd and Sharp40 found that poliovirus and reovirus would aggregate together when diluted. Aggregates were not dispersed in lake or distilled water buffer or phosphate buffered saline. Thus, it may be possible for human pathogenic viruses to form aggregates with the more abundant plant viruses41,42 and bacteriophages in wastewater, although this seems to not have been studied. This seems possible if they have similar isoelectric points in this environment.
Table 1. Factors Influencing Virus Aggregation virus structure (lipids, fibers) formed intracellularly during growth of the virus in the host. Can form as crystalline type structures (poliovirus) or grape like clusters associated with host cell debris resulting from destruction of the host cell when the pH of a solution approaches the isoelectric of the virion changes in the salt concentration or types of salts in solution natural organic matter present in water formed at solid surfaces including particulates in the water and flat surfaces polyelectrolytes induced formation action of a disinfectant (chlorine dioxide; UV light) repeated freezing and thawing
20, 31−34 8, 9 10−13 18 10, 20, 21 26−29 27−30 45 89, 91 36
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METHODS USED TO REDUCE AND ELIMINATE VIRAL AGGREGATES To understand the importance of viral aggregates several methods have been used to produce virus preparations free of aggregates or at least minimize their presence in suspensions (Table 2). One of the earliest techniques used was sonication. Figure 1. Physical state of virions and their aggregation inside host cells (A) cell lysates of infected cells (B) and in response to physical chemical conditions (C, D, E).
Table 2. Methods Used to Disperse Virus Aggregates method sonication differential density gradient centrifugation pH and salts
adenovirus20 and influenza22 in the presence of Ca2+ than Mg2+ has been observed. Anions in solution may also play a role in aggregate formation. MS2 coliphage was found to form aggregates of 300−400 nm in size when stored on phosphate buffered saline, but not when bicarbonate was used as a buffer.23 It was theorized that this was due to the phosphate linking of the amino acid lysine in the proteins of the virus coat resulting in the formation of aggregates. The association of viruses with particulate matter is known to enhance survival and resistance to disinfectants.24,25 However, they can also play a role in the formation of viral aggregates. The presence of natural organic matter (NOM) in surface water was found to enhance the formation of rotavirus aggregates especially in the presence of divalent cations.26 It also appears that viruses can form aggregates around/within particles by association with organic matter in surface waters.27−29 In this case, the organic flocs that form in surface waters act to bind together the viruses in a matrix. The presence of natural clays can also mediate viral aggregation.30 The
detergents freon or chloroform diethylaminoethyl dextran membrane filtration
virus vaccinia virus poliovirus type 1
reference 43 40
MS2 coliphage, poliovirus type 1, reovirus type 3, feline calicivirus poliovirus type 1
38, 40, 47 31, 47−49 poliovirus type 1, reovirus type 3, feline 49−51 calicivirus coxsackievirus B5 44 38
Almost 90% of vaccinia virus released from cell culture is in the aggregated form. Sonication was found to reduce this to 20%, but if the preparations were not diluted they quickly reaggregated.43 Jensen et al.44 found aggregates of coxsackieviruses very difficult to disperse and tended to aggregate even after separated by differential gradient centrifugation. Sodium chloride or pH manipulation had no effect on dispersion of the aggregates. Only the use of diethylaminoethyl was successful in 7319
DOI: 10.1021/acs.est.6b05835 Environ. Sci. Technol. 2017, 51, 7318−7325
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compliment reactivation was first described by Luria59 to account for infection of Escherichia coli by two or more bacteriophages exposed to ultraviolet light, which resulted in increased titer compared to infection by a single phage. MR has been demonstrated in cells of an organized host, including vaccinia virus,60 influenza virus,61 poliovirus,62 adenovirus63 and reovirus.64 The different reovirus types (T1L, T2J, T3D) have been demonstrated to undergo compliment reactivation among each other after exposure to UV light.64 MR was also observed in hydrogen-peroxide damages to the DNA of phage T4,65 thus MR may occur with chemical disinfectants as well as irradiation.
dispersion of the aggregates. To disperse aggregates formed in cell culture Freon or chloroform have been used extensively (Table 2). Grant45 and others26 have suggested that the concentration of salts, pH and human viruses are too low to form aggregates in natural aquatic environments and that aggregates formed in host cells are the only likely source. However, they did not consider the formation of aggregates with other viruses or the potential role of natural organic matter, which could also play a role.26−30
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SIZE OF VIRAL AGGREGATES Viral aggregates can occur in simple pairs to perhaps thousands of virions. Sharp et al.52 observed that large viral aggregates produced in cell culture were rarely found and not easily distinguished by electron microscopy and thus difficult to estimate their true numbers. Almost 90% of adenovirus 2 in cell culture preparations was observed to be aggregated by electron microscopy.38 An estimated 57% of the aggregates contain 26 or more virions and 35% ranged from 51 to 150 virions. In cell culture preparations of vaccinia virus, 69 to 90% of the virus was found to be aggregated.53 Aggregates of up 150 virions were observed, but most were in the 2−28 virion size range. Echovirus type 4 cell culture preparations were filtered through different sized membrane filters, treated with serum to prevent virus adsorption, prior to determine the size of aggregates.54 It was found that 30% of the viruses were aggregated and that 9% were greater than 450 nm in diameter. They also reported that treatment of the preparation with trypsin eliminated most of the aggregates. Virus can be made to aggregate by adjustment of the pH or salt concentration and type of cationic salt in suspension. Mattle et al.47 created aggregates of MS2 coliphage (23 nm in diameter) by lowering the pH to near the virus’s isoelectric point (pI = 3.9). The rate of aggregation was dependent upon the pH. After 1 h at pH 3.0, the radius of the viral aggregates was 1,000 nm, whereas at pH 3.6 the radius was 750 nm. Aggregates can also be induced to form around clays and likely other types of particulate matter. Aggregates of ∼100 influenza virions (2,000 μm in size) were found to form in the presence of montmorillonite and remain infectious in cell culture and act as a single infectious unit.30 Cationic organic molecules can also induce the formation of viral aggregates. Pinto et al.55 found that polyhexanethylene biguanide cationic disinfectant could induce the formation of aggregates changing the disinfectant inactivation kinetics, thus overestimating the effectiveness of the disinfectant. Using light scattering they showed that aggregates up to 500 nm of MS2 coliphage were formed, which effectively decreased the number of plaque forming units of the virus.
DETECTION AND QUANTIFICATION OF VIRAL AGGREGATES Technical progress in the field of microscopy and the adaptation of applications originally developed for use in nanotechnology spurred the quantitative analysis of virus particles as physical entities.66 Field flow fractionation connected with multiangle light scattering (FFF-MALS), dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), tunable resisting pulse sensing (TRPS) size exclusion chromatography coupled with MALS (SECMALS), disc centrifuge sedimentation (DCS) and analytical ultracentrifugation (AUC) are technologies available for determination of virus particle concentration and characterization including virus integrity, size and physical or aggregation state.66−71 Full description of methods to determine virus particle concentration and extent of aggregation have been reported for different viruses and biotechnology applications.66,68,70,72−75 No single technique is able to directly quantify incomplete viral particles and aggregates at the same time.75 Virus characterization, sizing and concentration measurement have been determined for virus suspensions absorbed onto carbon-coated electron microscopy grids using highresolution electron microscopy, for example, atomic-resolution transmission electron microscopy (TEM).38,47,76 In addition, atomic force microscopy (AFM) has been used to visualize virus structure with images and information that are distinct from those obtained by electron microscopy, and in some cases, at even higher resolutions. AFM produces three-dimensional, topological images that accurately depict the surface features of the virus. The images resemble common light photographic images and require little interpretation. The structures of viruses observed by AFM are consistent with models derived by X-ray crystallography and cryo-EM.77−79 Laser-based nanoparticle tracking analysis (NTA) systems have been employed for high throughput analysis of total virus particles and level of virus aggregation. NTA allows direct visualization of virus particles in suspension and provides absolute concentration measurement in virus particle per milliliter. Particles sizes ranging from about 30 to 1000 nm and particle concentrations ranging from 107 to 109 particles/ mL, depending on sample type, can be analyzed by NTA. This technique combines laser light scattering microscopy with a charge-coupled device (CCD) camera operating at 30 frames per second which enables the visualization of the Brownian motion of virus particles in liquid.66,80 More recently a complementary metal−oxide−semiconductor (CMOS) sensor based system which transforms the optical information into electronic data enables more frames per second. The video captured with the camera showing the movement of individual particles can be subsequently analyzed using NTA software.66 Lower size limits for this detection method range between 10
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INFECTIVITY AND COMPLIMENT REACTIVATION Virus aggregates will produce individual plaques in cell culture or in the case of bacteriophages on bacterial lawns of the host bacterium identical to those observed for individual virions.53,56−58 Thus, this can result in an underestimation in the true number of infectious virions. Multiplicity reactivation (MR) involves the infection of a cell by the cooperative effort of two or more UV and Gamma irradiated-damaged viruses, none of which is completely functional alone.59−61 This phenomenon also known as 7320
DOI: 10.1021/acs.est.6b05835 Environ. Sci. Technol. 2017, 51, 7318−7325
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Figure 2. Inactivation rates of poliovirus preparations (poliovirus type 1, Mahoney strain) containing single, small aggregates, and large aggregates exposed to bromine8 Virus inactivation decreases over time due to viral aggregates.
Table 3. Impact of Aggregates on Increase of Resistance to Disinfectants virus
disinfectant
increase in resistance
remarks
reference
adenovirus poliovirus 1 poliovirus feline calicivirus hepatitis A rotavirus SA 11 rotavirus SA11 MS2 coliphage rotavirus SA11 hepatitis A MS2 coliphage MS2 coliphage generic generic MS2 coliphage
chlorine chlorine chlorine chlorine chlorine chlorine chlorine dioxide chlorine dioxide monochloramine monochloramine peracetic acid dichloramine generic UV light UV light
increase in Ct up to 3-fold increase of 2−2.5 at pH 6 2-fold 31 to 422-fold increase in Ct at pH 7 12.6-fold at pH 6.0; 5.4-fold at pH 10.0 10-fold increase in Ct at pH 6; 2.5-fold pH 10 5.2-fold at pH 6; ≥ 1.6 at pH 10 average 21-fold (range 5 to 95-fold) at pH 6.5 1.5-fold at pH 8 1.42-fold at pH 8.0 2 to 6-fold 3.4 to 13-fold pH 3.0 4-fold 10% increase in first order inactivation rate constants 2-fold
induced by pH cell culture produced cell culture produced cell culture produced cell culture produced cell culture produced cell culture produced induced by the disinfectant cell culture produced cell culture produced induced by pH induced by pH based on a kinetic model based on a model from the data by Mattle et al. 2011 induced by pH and salts
38 51 8 51 86 87 87 88 87 86 47 35 46 94 91
aggregates were nonlinear and required twice the time for the same degree of inactivation as monodispersed preparations8 (Figure 2). Interestingly some preparations of aggregated virus were inactivated in a linear fashion, although at a lower rate than monodispersed preparations. The authors suggested that the linear rate of virus inactivation was probably due to the right proportion of aggregate sizes in the virus preparations. Pancorbo et al.82 found that preparations of heavily aggregated human rotavirus strain Wa were more resistant to free chlorine than nonaggregated simian rotavirus. Cell associated aggregates of poliovirus, hepatitis A and feline calicivirus have also been found to be highly resistant to chlorine disinfection.51,86 The Ct for a 2−4 log decrease for feline calicivirus during disinfection with free chlorine at pH 7 was found to be 31−442 times greater for a 4-log reduction for cell associated aggregates vs Freon dispersed virus. In another study, the Ct for a 4-log reduction of cell associated hepatitis A virus was found to be 13.6-fold greater at pH 6. Virus reduction was 5.4-fold greater at pH 10 where the virucidal effect of free chlorine decreases due to the predominant occurrence of hypochlorite ion (OCl−) which is a weaker disinfectant than hypochlorous acid (HOCl). The difference was even less in the presence of chloramine (a 1.4-fold difference). Similar results
nm for particles with high refractive index (Ri) such as colloidal gold, and 30 nm for lower Ri such as particles with biological origin (i.e., viruses).
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IMPACT ON SURVIVAL IN WATER The state of virus aggregation should be taken into consideration when assessing virus survival in water under laboratory conditions. Gassilloud and Ganzter81 found that after 20 days of studying poliovirus type 1, 8 to 26% of the decrease in virus titer was due to virus aggregation. Previous studies attributed the greater survival of human rotavirus Wa over simian rotavirus SA-11 due to the ability of the formers ability to generate large aggregates in cell culture preparations as revealed in electron micrographs.82 Aggregates may also increase the apparent thermo-stability of viruses.83
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IMPACT ON DISINFECTION Berg et al.84 first suggested that observed decreases in inactivation rates of poliovirus by a disinfectant over time could be due to the presence of aggregates. During the 1970s studies were conducted to better define the inactivation kinetics of aggregates by chlorine and bromine disinfection.8,52,85 These studies indicated that preparations of poliovirus containing 7321
DOI: 10.1021/acs.est.6b05835 Environ. Sci. Technol. 2017, 51, 7318−7325
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have been found for rotavirus SA-11 with chlorine, monochloamine and chlorine dioxide87 (Table 3). This pattern fits the suggestion of Mattle et al.47 that less reactive disinfectants would be more effective against viral aggregates. Disinfectants may also impact the formation of aggregates. Chlorine dioxide was found to promote the formation of aggregates of MS2 coliphage88 through particle destabilization, but not chlorine.89 The formation of the aggregates increased the resistance of the phage to chlorine dioxide by an average of 21 times. Grant46 developed a kinetic model for the inactivation of viral aggregates that compared well with the observed data. The model predicted that viral aggregates would be about four times more resistant than individual virions to disinfectants (Table 3). Mattle et al.47 conducted a detailed study on the inactivation of MS2 coliphage aggregates induced by lowering the pH of a solution containing peracetic acid (Table 3). Aggregation was found to reduce the inactivation rate constants 2−6-fold. Genicord35 found that the larger the aggregates of MS2 coliphage the greater the resistance to inactivation by dichloramine. Thus, the larger the aggregate the greater the time needed for inactivation of the viruses. Using a rate diffusion model, it was found that the inhibitory effect of aggregation resulted from consumption of the disinfectant within the aggregate, but that the diffusion of the disinfectant into the aggregates was not a limiting factor. From this observation, the authors suggested that aggregates would be more resistant to more reactive disinfectants, such as chlorine. Thus, they suggested that milder disinfectants should be used in water containing vial aggregates. Mattle and Kohn90 continued their studies with MS2 coliphage aggregates during UV light disinfection. In this study, the virus aggregates were dispersed after UV light treatment before assay. They found that the initial inactivation kinetics were similar for viruses incorporated into the aggregates (up to 1000 nm in radius) than dispersed viruses. However, aggregated viruses started to tail more readily than the dispersed ones. They concluded that neither light shielding nor the presence of resistant subpopulations was responsible for this tailing effect, but was due to recombination of virions fused by the exposure to UV light. In contrast, Feng et al.91 found that aggregates of MS2 coliphage induced by pH and CaCl2 concentration were more resistant to UV by about 2fold. The inactivation of aggregated vaccinia virus by UV light was found to be nonlinear compared to monodispersed preparations.92 Vaccinia virus contains a lipid envelope and a linear, double-stranded DNA genome. The virus used in this study was produced in cell culture from which the aggregates originated. They were not produced by manipulation of pH or ionic concentration. Such aggregates may contain particulate organic matter originating from cell debris that may aid in protecting the virus from inactivation by UV light. A virus sensitivity index or VSI as a means of quantitatively evaluating parameters influencing UV light disinfection was developed by Tang and Sillanpäa.̈ 93 This index was defined as the ratio between the first-order inactivation rate constant of a virus and that of MS2 coliphage during UV light disinfection. Their model indicated that aggregates of 500 nm would reduce the rate constant by 10% for human enteric viruses. However, this assumes that naturally occurring enteric virus aggregates behave in the same manner as laboratory induced aggregates of MS2 coliphage.
Critical Review
FILTRATION
Membranes used in ultrafiltration and reverse osmosis are part of the treatment train for advanced water treatment processes designed to reduce chemical and microbial contaminates.95−98 The efficacy of these membranes for virus removal is assessed by challenging them with viruses.99−101 If aggregates are present in the virus preparation used in these studies an overestimation of virus removal may result. Langlet et al.94 studied four different phages during removal by membrane filtration. They found that aggregate formation of the viruses was dependent upon the nature of the virus, pH and ionic strength of the virus preparation. However, it was not predictable for each virus. Qβ phage aggregation occurred when the pH decreased in addition to increasing ionic strength, while phages SP and GA aggregated over the entire range of pH-ionic strength conditions tested. They suggested that MS2 and Qβ phages at neutral pH and low ionic strength met the right criteria for the viruses to express a negative charge. During reverse osmosis MS2 coliphage is extensively aggregated in the membrane filter retentate due to the high salt concentration.102 Thus, an infectivity assay would underestimate the level of viruses present. Larger pore sized microporous membrane filters in the millimicron range can also retain large aggregates.103 Gutierrez et al.104 found that little aggregation of a porcine rotavirus would occur at salt concentration and pH conditions of groundwater and suggested that aggregation of this virus would not be a factor in deposition or transport through groundwater.
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CONCLUSIONS Virus aggregation-disaggregation is a complex process and predicting the behavior of any individual virus is difficult under a given set of environmental circumstances without actual experimental data. It is clearly very virus specific, making generalizations difficult because of differences in the chemical and physical structure of viruses by type and even strain. Animal viruses produced in cell culture form crystalline like structures and form aggregates that are often associated with cell debris and other organic matter present in feces. These aggregates may behave very differently than those formed in suspension by manipulation of the pH and salt concentration in the laboratory. They may be more difficult to disperse and the presence of organic matter may make them more resistant to certain disinfection processes because of the increased demand by the disinfectant (e.g., free chlorine). For these reasons data collected on the behavior of coliphage aggregates may not reflect those formed by those produced in cell culture. Recent insights also indicate that lower reactive disinfectants may be better in inactivating virions within the aggregate. Additional basic research on viral aggregates will help optimize disinfection of water and wastewater systems, and provide insights into why some virus types are more resistant than others to disinfection.
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AUTHOR INFORMATION
Corresponding Authors
*(C.P.G.) E-mail:
[email protected]. *(W.Q.B.) Phone: (520)-621-6163; e-mail: wbetancourt@ email.arizona.edu. ORCID
Walter Q. Betancourt: 0000-0003-0488-4483 7322
DOI: 10.1021/acs.est.6b05835 Environ. Sci. Technol. 2017, 51, 7318−7325
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Environmental Science & Technology Notes
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The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This review was supported in part by the United States Department of Agriculture-National Institute of Food and Agriculture. Grant number 20166800725064, that established CONSERVE: A Center of Excellence at the Nexus of Sustainable Water Reuse, Water and Health. We wish to thank Mr. Jeffrey R. Bliznick for providing the graphics included in this review.
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