Inactivation and Tailing during UV254 Disinfection of Viruses

Aug 22, 2012 - conclusively demonstrated. This study investigates how aggregation affects virus inactivation by UV254 in general, and the tailing phen...
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Inactivation and Tailing during UV254 Disinfection of Viruses: Contributions of Viral Aggregation, Light Shielding within Viral Aggregates, and Recombination Michael J. Mattle and Tamar Kohn* Laboratory of Environmental Chemistry, School of Architecture, Civil and Environmental Engineering (ENAC), École Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland S Supporting Information *

ABSTRACT: UV disinfection of viruses frequently leads to tailing after an initial exponential decay. Aggregation, light shielding, recombination, or resistant virus subpopulations have been proposed as explanations; however, none of these options has been conclusively demonstrated. This study investigates how aggregation affects virus inactivation by UV254 in general, and the tailing phenomenon in particular. Bacteriophage MS2 was aggregated by lowering the solution pH before UV254 disinfection. Aggregates were redispersed prior to enumeration to obtain the remaining fraction of individual infectious viruses. Results showed that initial inactivation kinetics were similar for viruses incorporated in aggregates (up to 1000 nm in radius) and dispersed viruses; however, aggregated viruses started to tail more readily than dispersed ones. Neither light shielding, nor the presence of resistant subpopulations could account for the tailing. Instead, tailing was consistent with recombination arising from the simultaneous infection of the host by several impaired viruses. We argue that UV254 treatment of aggregates permanently fused a fraction of viruses, which increased the likelihood of multiple infection of a host cell and ultimately enabled the production of infective viruses via recombination.

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INTRODUCTION Disease outbreaks originating from contaminated drinking water continue to pose a health risk even in countries with sophisticated water infrastructure. While major diseases of the past, such as cholera and typhoid fever, have greatly diminished, they have been replaced by outbreaks caused by chlorineresistant pathogens such as Cryptosporidium or emerging pathogens including protozoa, bacteria, and viruses.1,2 Inadequately applied disinfection techniques, disinfectant concentrations, and treatment times allow resistant pathogens to remain infective in drinking water, or to regrow in the distribution system. These microorganisms can thereafter infect consumers and cause a variety of diseases, ranging from simple diarrhea to more serious illnesses.3 UV disinfection is an increasingly popular water treatment technique; in Europe it has been widely used for decades.4 This disinfection method has recently received renewed interest mainly for two reasons: first, it was shown to be effective against (oo)cysts of Cryptosporidium5,6 and Giardia,7 organisms © 2012 American Chemical Society

that are highly resistant to chlorine treatment. Second, UV disinfection avoids the production of carcinogenic byproducts which form during chlorine disinfection.8,9 Viruses and spore-forming bacteria are among the most resistant waterborne pathogens toward UV radiation.10−12 They are therefore good indicator organisms to determine the fluence needed to obtain safe drinking water. UV254 disinfection of viruses has frequently been reported to exhibit an initial exponential decrease in infective viruses, followed by tailing.10,11,13−16 A subset of viruses thus appears to be protected from the effects of UV254 disinfection, and may jeopardize achievement of the treatment goal. Different hypotheses have been put forward to explain the tailing phenomenon, including light shielding by particles or viral Received: Revised: Accepted: Published: 10022

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all buffers were filtered with a cellulose nitrate membrane filter (0.1 μm pore size; Whatman GmbH, Dassel Germany). The samples were then left to aggregate under constant stirring. Aggregate formation was monitored by periodically withdrawing 70-μL aliquots which were immediately analyzed by DLS (for details see SI). After 30 min, aggregates of 800 and 1000 nm in radius had formed at pH 3.6 and 3.0, respectively. At higher pH values, the samples remained dispersed. Additionally, experiments were performed at a lower virus concentration (5 × 108 pfu/mL), which yielded smaller aggregates, namely around 100 nm in radius at pH 3.0. Following UV254 treatment, viruses were redispersed by raising the solution pH to 7.0. This was accomplished by the addition of phosphate-buffered saline (PBS: 5 mM phosphate, 10 mM NaCl, pH 7.5). Control experiments were performed in the absence of UV254 irradiation to study the dispersion efficiency upon increasing the pH, as well as the stability of viruses at pH 3.0. A 0.1−0.5 log10 loss in infectivity was observed in samples that were aggregated at pH 3.0 and then redispersed. Over the time frame of the disinfection experiments at pH 3.0 a 0.0−0.4 log10 loss was observed. Disinfection by UV254 Irradiation. Experiments were conducted at 22 °C in 15 mM phosphate and 15 mM chloride at pH 3.0 and 3.6, and 4.0, 4.5, and 5.0, for aggregated and dispersed viruses, respectively. Two mL of buffer solution was placed into a 5-mL beaker and continuously stirred at 200 rpm. Twenty μL of a 5 × 1012 or 5 × 1010 pfu/mL virus stock solution (in 10 mM phosphate and 5 mM NaCl, pH 7.5) was added to the buffer and left to aggregate for 30 min before UV254 irradiation was started. Aggregate sizes (z-average values) were determined by DLS immediately before UV254 irradiation was started and after the experiments were concluded. A low-pressure 18 W UV−C lamp (253.7 nm, model TUV T8 Philips) in a quasi-parallel beam setup as proposed by Bolton and Linden28 was used to inactivate the phages. To optimize beam collimation, the 5-mL beakers, painted black, were placed in black plastic tubes (3 cm diameter and 17.5 cm high). The fluence entering the reactors was measured by actinometry29 to inactivation experiments. For each experiment duplicate samples or more were run in parallel. Sample aliquots of 100 μL were extracted periodically, diluted in 350 μL of PBS, and enumerated immediately upon conclusion of the experiment. A set of control experiments was performed in 400 mM phosphate and 15 mM chloride, which prevented virus aggregation over the whole pH range considered.26 This allowed us to investigate the effect of pH on the experimental system without the confounding effect of simultaneous aggregation. For enumeration, these samples were diluted using high-concentration phosphate-buffered saline (150 mM phosphate, 15 mM chloride at pH 7.5). Quantitative Polymerase Chain Reaction (qPCR). The UV254-induced genome damage was measured by qPCR as described in detail elsewhere.30 A primer set (forward sequence: CCGCTACCTTGCCCTAAAC; reverse sequence: GACGACAACCATGCCAAAC) was chosen that amplified a genome segment of 303 nucleotides (657−959). As described previously,30 the intact proportion of this genome segment after UV254 treatment (n/n0), determined by qPCR, was extrapolated to obtain the intact proportion of the whole genome (N/N0) as follows: N/N0 = (n/n0)c where c is 10.1.

aggregates in solution,14 or the presence of resistant virus subpopulations;17 however, no conclusive evidence is available to support these hypotheses.12 Other studies simply state that tailing is due to virus aggregation, without any further discussion of how aggregation would lead to such an effect.18−20 A possible explanation for the effect of virus aggregation on UV254 disinfection was first put forward by Sharp and coworkers21,22 who inactivated dispersed and aggregated samples of vaccinia viruses. The aggregated samples were found to be more resistant to UV254 radiation than the dispersed ones. On a semilog scale (log of infective concentration vs fluence), aggregates mostly exhibited a tailing curve, though occasionally linear inactivation behavior was also observed. They concluded that the tailing feature could not be attributed to multiple hits needed to inactivate all the viruses within an aggregate. Instead, the reduced inactivation rates were consistent with recombination, a theory first described by Luria and Dulbecco.23 In brief, this theory reports that a cell infected by several genomedamaged viruses can regenerate infective viruses with an intact genome. To do so, the polymerase switches from one genome strand to another during the replication process.24,25 Therefore, an integral genome can be created by using the intact portions of the genomes of several damaged viruses as templates. Contributions of light shielding by aggregates or the presence of resistant subpopulations, however, were not considered in this study. The aim of this work was to elucidate the origin of the tailing effect during UV254 disinfection of viruses. In particular, the effect of virus aggregation (i.e., the conglomeration of multiple viruses) with respect to light shielding and recombination was considered. We developed an experimental approach that allowed us to compare disinfection of dispersed viruses with that of viral aggregates of well-defined size. Aggregates were redispersed after treatment and before enumeration. In contrast to previous studies, it was therefore possible to study the effect of UV254 treatment on single viruses within an aggregate rather than the inactivation of an aggregate as a whole. The results from our study thus remain valid even if viral aggregates disperse in post-treatment environments.

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EXPERIMENTAL SECTION Dispersed and aggregated samples of bacteriophage MS2 were irradiated with UV254 and the inactivation kinetics were monitored. MS2 was first aggregated by reducing the solution pH, then aggregates were inactivated by UV254, redispersed, and finally the residual infective viruses were enumerated. Dynamic light scattering (DLS) was employed to determine aggregation size before and after UV254 treatment. Chemicals and Organisms. All chemicals, as well as all microorganisms and their associated culturing, purification, and enumeration methods are described in the Supporting Information (SI). Aggregate Size Measurements. Aggregate size was measured by dynamic light scattering (DLS) and transmission electron microscopy (TEM) as described previously.26 The detailed experimental methods are described in the SI. Viral Aggregation and Dispersal. Aggregation was initiated by lowering the solution pH below the MS2 isoelectric point (pI) of 3.9.27 A solution containing 5 × 1010 infective viruses/mL (enumerated as plaque-forming units (pfu)/mL) was placed into a 15 mM phosphate and 15 mM chloride buffer at different pH values of 3.0, 3.6 4.0, 4.5, and 5.0. Prior to use, 10023

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Figure 1. UV254 disinfection performed in 15 mM phosphate and 15 mM chloride (a) at pH 5.0 (dispersed viruses) and (b) at pH 3.0 (aggregated viruses), at an initial MS2 concentration of 5 × 1010 pfu/mL. Different symbols indicate four replicate experiments. The k values in (a) varied from 0.044 to 0.052 cm2 mJ−1. The average aggregate radius in (b) was 924 ± 191 and k values varied from 0.114 to 0.137 cm2 mJ−1. Arrows indicate that the remaining infective virus concentrations were below the detection limit. The inset represents the averaged fraction of intact genome copies (N/ N0) of two experiments (red and green).

Correction for Light Attenuation and Scattering. The fluence determined by actinometry reflects the total intensity of the incoming light. In our experimental solutions, this light was absorbed by viruses as it passed through the reactor. Therefore, the UV254 intensity in solution decreased with increasing reactor depth. The average fluence throughout the reactor was calculated by multiplying the incoming fluence with a light attenuation factor S(λ)28,31 according to Morowitz,32 assuming a perfectly mixed solution. S (λ ) =

1 − 10−α(λ)l α(λ) ·l·ln(10)

and in part due to backscattered light not detected by the integrating sphere. The difference in α between the aggregated (pH 3.0) and dispersed (pH 5.0) samples corresponds to the total light scattered by the aggregates. In our experimental setup the two values of α differed by 38%. According to Beer−Lambert law, for our sample depth of 0.64 cm, this corresponds to 7% of the incoming light being scattered. As this value is low, the effect of light scattering was neglected in this work. Therefore the same light attenuation factor S(λ) was used for both dispersed and aggregated samples. Data Analysis. Inactivation data that exhibited exponential decay over time were fitted to a log10-based first order inactivation model

(1)

where α(λ) is the decadic attenuation coefficient (cm−1) and l is the path length of the light in solution. This correction is applicable as long as there is no significant light scattering due to particles in solution. To determine if viruses and viral aggregates scattered light, the UV absorbance at 254 nm of dispersed and aggregated samples was measured using a UV−vis spectrophotometer (UV-2550, Shimadzu), and compared to measurements using the same spectrophotometer but with an integrating sphere attachment. The integrating sphere (ISR-240A, Shimadzu) collects all the forward scattered light and corrects the absorbance value to reflect only true light absorption, assuming that no backscattering occurs. Absorbance measurements of dispersed virus samples at pH 5.0 were comparable when determined with and without the integrating sphere (αIS = 0.079 cm−1 and α = 0.080 cm−1 with and without the integrating sphere, respectively). This indicates that light scattering was negligible in the dispersed samples. When the pH was lowered to 3.0 and the viruses aggregated, αIS and α corresponded to 0.098 and 0.128 cm−1, respectively. The difference between these two values was attributed to light scattering by the viral aggregates. Furthermore, αIS obtained at pH 3.0 was larger than at pH 5.0. This increase could not be explained by enhanced light absorption by the viruses: absorption by RNA at 254 nm decreases with decreasing pH due to the protonation of cytosine,33 and the absorption by viral proteins was negligible (see SI). Instead, the larger value αIS obtained at pH 3.0 is in part due to the increased path length (and hence increased light absorption) due to scattering,

⎛c ⎞ log10⎜ t ⎟ = −k· fluence rate ·t ⎝ c0 ⎠

(2) 2

−1

where k is the inactivation rate constant (cm mJ ), c0 (pfu/ mL) is the initial infective MS2 concentration, and ct is the remaining infective MS2 concentration after irradiation time t (s). The fluence rate (mWcm−2) multiplied by t equals the fluence or UV254 dose (mJ cm−2). The last point used for the regression of the initial exponential decay was chosen such that the lowest possible 95% confidence interval on the slope was obtained. The k values are reported as the mean of all replicate experiments. The associated error reflects the highest and lowest value measured.

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RESULTS AND DISCUSSION Effect of Aggregation on Inactivation. UV254 disinfection led to an initial exponential decay of infective viruses for both dispersed viruses and aggregates. However, at higher fluences the inactivation curves started to tail (Figure 1). Because the viral aggregates were redispersed before enumeration, the data describe the inactivation of single viruses that were incorporated in aggregates during UV254 treatment. At pH 5.0, a k value of 0.047 cm2 mJ−1 (0.043−0.052 cm2 mJ−1 for 9 replicates) was determined for the exponential part of the inactivation curve. This corresponded well to reported literature data.12,14,34,35 The reduction of the solution pH to 3.0 increased the k value by a factor of 2.4 to 0.113 cm2 mJ−1 (0.097−0.137 cm2 mJ−1 for 9 replicates). As discussed in the 10024

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3.0), but the virus concentration was lowered a hundred-fold. This led to smaller average aggregates sizes (∼ 100 nm as compared to ∼1000 nm for high virus concentration) and thus allowed a direct comparison of light shielding between large and small aggregates. If tailing were due to light shielding, the smaller aggregates should exhibit a later onset of tailing than the large ones. Results showed first that the aggregate size did not affect the exponential part of the inactivation curves; the difference between the k value of small and large aggregates was negligible (SI Figure S2). This confirms that aggregation had little impact on k. Similarly, the onset of tailing occurred between 4 and 6 log10 of inactivation for both small and large aggregates (Figures 1b and SI S3). This indicates that the tailing effect was not due to light shielding, as smaller aggregates should be less efficient at protecting the viruses at the core of the aggregates from UV254 rays. Further confirmation that aggregates did not cause significant light shielding was obtained by monitoring the decay of the MS2 genome. Genome degradation during UV254 inactivation of aggregated and dispersed viruses is shown in the insets of Figure 1. It can be seen that in both cases genome degradation followed first-order kinetics throughout the entire experiment; even at higher fluences no tailing was observed, while the loss in infectivity leveled off. This demonstrates that UV254 rays could access all genomes with the same efficiency, independent of their location in an aggregate. Furthermore, it suggests that virus inactivation in the tailing part of the experiments was not proportional to genome damage, a finding that will be further discussed below. Modeling Light Shielding within Viral Aggregates. To evaluate if light shielding can be important for aggregate sizes or viruses not studied herein, we developed a general model that captures the effect of aggregation on disinfection by light. The model needed to couple two processes: the penetration of light into the aggregates and the inactivation of viruses due to the local light intensity. The average concentration of infective viruses within an aggregate, cv , for any given fluence, virus attenuation coefficient α(λ), and inactivation rate constant k(λ) were computed using eq 3 (the full derivation is reported in the SI)

Experimental Section, UV254 absorption of the RNA does not increase at lower pH and thus cannot explain the enhanced inactivation rate. However, at pH 3.0 the amine groups of adenosine and cytosine become protonated.36 This reduces the overall negative charge of RNA, and decreases the repulsive forces between negatively charged entities (note that the pI of the MS2 RNA was estimated to be 2.9,37 thus the overall charge of RNA should be close to zero in our experimental solution of pH 3.0). This could lead to a more densely packed genome, which is more susceptible to UV254 inactivation,19 as it may act as a sensitizer for its own degradation. To determine if the different k values obtained in dispersed and aggregated samples were only due to the different sample pH, or if aggregation played a role, they were compared to a second set of k values obtained at a phosphate concentration of 400 mM. Under these conditions, viruses remain dispersed at both pH 5.0 and 3.0.26 This system thus allowed a direct comparison of dispersed and aggregated viruses at a single pH. The increased phosphate concentration had a negligible impact on the disinfection kinetics at pH 5.0 (Figure 2). By decreasing

Figure 2. Inactivation rate constants k versus pH for experiments performed in 15 mM phosphate and 15 mM chloride (blue bars) or 400 mM phosphate and 15 mM chloride (red bars), at an initial MS2 concentration of 5 × 1010 pfu/mL. The average aggregate radius was 955 ± 199 at pH 3.0 and 15 mM phosphate. All other samples contained dispersed viruses. Bar heights indicate the mean values for n replicates. Error bars indicate lowest and highest k value measured.

cv(fluence, α(λ), k(λ)) R 3 = 3 r 2cv ,010−k(λ)·S(r , α(λ))·fluencedr R 0

the pH to 3.0, the k value increased dramatically for both low and high phosphate concentrations. The aggregated samples showed a slightly lower average k value than the dispersed ones; however, the difference was much smaller than the experimental error (Figure 2; for a detailed discussion of the effects of phosphate at pH values between 3.0 and 5.0, see SI). Even if correcting for the fact that light was scattered to a greater extent in the aggregated samples, scattered light would account for no more than a 7% decrease in k (see Experimental Section). Overall, these results show that the increased k in aggregated samples at pH 3.0 could be attributed to a pH effect. Aggregation, however, had little impact on the initial exponential decay. In contrast, aggregation did affect the onset of tailing, which occurred more readily in aggregated samples (Figure 1). Effect of Light Shielding on Inactivation of Aggregates. To determine if the earlier onset of tailing was due to light shielding within the aggregates, a second set of experiments was performed in which the buffer concentration and pH were kept constant (low phosphate concentration, pH

∫

(3)

where S(r,α(λ)) is the average light shielding at distance r from the core of the aggregate (see Figure S4). Inactivation of MS2 aggregates by UV254 was modeled using an α(254 nm) of 4430 cm−1 (see SI for details) and the k(254 nm) value of 0.116 cm2 mJ−1 was determined experimentally for dispersed viruses at pH 3.0 in 400 mM phosphate (Figure 2). Light shielding led to a slight decrease in inactivation, depending on the aggregate radius (Figure 3). k was reduced by 53% for aggregates with a radius of 1000 nm compared to dispersed viruses. Additionally, the model exhibited only a very mild tailing, even for the largest modeled aggregates (Figure 3). This confirms that the strong experimentally observed tailing (Figure 1b) could not be attributed to light shielding within viral aggregates. A comparison of the modeled and experimental data showed that the modeled data overpredicted the experimentally determined effect of light shielding. This discrepancy can be 10025

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Figure 4. Modeled inactivation curves for different viruses taking into account light shielding within aggregates of 500 nm in radius (solid lines), compared to inactivation of the corresponding dispersed viruses (dashed lines). The viruses presented in this graph are adenovirus (light green), calicivirus (blue), poliovirus (orange), and MS2 (light blue). For disperse viruses, reported k values of 0.024, 0.106, and 0.135 cm 2 mJ−1 were employed for adeno-, calici-, and poliovirus, respectively;12 for MS2, the k of 0.047 cm2 mJ−1 determined herein (pH 5.0) was used. The calculated α(254 nm) values were 1900, 3360, 7000, and 4430 cm−1, respectively. The modeled k values for aggregates corresponded to 0.021, 0.080, 0.073, and 0.033 cm2 mJ−1, respectively.

Figure 3. Modeled inactivation curves taking into account light shielding within aggregates of 100 nm (light blue ), 250 nm (orange ), 500 nm (light green ), and 1000 nm (blue ) in radius compared to dispersed viruses (black −). Measured inactivation of large aggregates ■: k = 0.103 ± 0.009 cm2 mJ−1, radius 1030 ± 45 nm; ●: k = 0.107 ± 0.005 cm2 mJ−1, radius 1062 ± 172 nm. The calculated k values for aggregates were 0.108, 0.097, 0.082, and 0.058 cm2 mJ−1 for 100, 250, 500, and 1000 nm in radius, respectively.

attributed to a variety of reasons: the viral aggregates were not perfectly spherical in shape, which reduced the average light shielding as the light had shorter travel paths than calculated by the model; the packing of the viruses was assumed to be maximal, yet void spaces within viral aggregates had been observed on TEM images; the presence of empty viral capsids in the sample could reduce the actual amount of light shielded by the aggregate; and finally, the z-average values reported here as the average aggregation sizes were likely an overestimation of the real average radii in solution (see SI for details). To evaluate the light-shielding potential within viral aggregates for other viruses, attenuation coefficients were calculated based on the respective viral diameter, genome length,38 and attenuation coefficients of DNA and RNA39 (see SI for details), and reported k values12 were employed. The main influence on light shielding stems from the compactness of the viral genome within the capsid. Poliovirus, for instance, is only slightly larger than MS2 (30 nm diameter); however, it has a genome, which is twice as long. The more densely packed genome results in more important light shielding within an aggregate of 500 nm radius (Figure 4). Alternatively, calicivirus has a similar genome length as poliovirus but is slightly larger (38−40 nm diameter). This resulted in less important light shielding. The maximally observed difference in k between dispersed viruses and viruses in aggregates of 500 nm radius occurred for poliovirus and corresponded to a factor of 0.54. For the other viruses investigated, the difference in k was smaller. These results thus indicate that during water treatment by UV254 light shielding within viral aggregates should hardly ever be of importance. However, many wastewater particles have higher attenuation coefficients than determined here for viruses,40 and light shielding has been shown to arise from the adsorption of viruses onto nonviral particles that strongly absorb UV254.41 The lack of susceptibility of k to aggregation during UV254 disinfection is in stark contrast to disinfection by chemical disinfectants. In particular for strong oxidants, viral aggregates can have an important effect on k. Unlike UV254 intensity, the concentration of oxidants was found to decrease toward the core of the aggregates, and hence inactivation became less efficient with increasing aggregate size.26

Tailing Effect Is Consistent with Recombination of Viral Genomes. With light shielding eliminated as the cause of tailing, two other options remain: the presence of subpopulations with differing susceptibilities to UV254, or recombination. The former cause is unlikely, as the reculturing of the tailing MS2 population did not show enhanced resistance to UV254 (personal communication Ch. Gantzer). In addition, the significantly earlier onset of tailing at pH 3.0 compared to pH 5.0 in experiments using the same virus stock solution (Figure 1) further contradicts the theory of a resistant subpopulation. It should futhermore be noted that while many bacteria42−44 or double-stranded DNA viruses can use cellular machinery to repair UV-induced damage,13,35 no such mechanism is known for single-stranded RNA viruses such as MS2. Resistance arising from post-treatment repair thus cannot explain the observed tailing effect. The remaining explanation for the observed tailing effect is thus recombination, which has the potential to regenerate infective viruses by combination of several impaired viruses.22,23,45 Interestingly, pyrimide dimers formed due to UV disinfection of DNA viruses were shown to increase the frequency of recombination.46 For RNA viruses, recombination was first discovered for poliovirus,47,48 and it is now known to be common for animal, plant, and bacterial viruses,25,49 including MS2.50 For recombination to occur, several inactivated MS2 viruses have to infect the same host cell. Multiple infection of a host cell would likely be facilitated if the viruses were present as clumps containing several viruses. For example, Abel51 reported that recombination of vaccinia virus in large host cells occurred only if the viruses were preclumped with MgCl, and a similar observation was reported for poliovirus.52 The formation of small, non-dispersible clumps was also observed in our experimental system. As discussed in the Experimental Section, the redispersion of aggregates prior to enumeration was typically efficient. Consequently, samples which were aggregated at pH 3.0 and were redispersed exhibited a DLS-measured radius (z-average value) which was 10026

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of protein fusion upon UV254 treatment,53,54 and was confirmed by TEM analysis, which revealed an increase in viral clumps containing two or more viruses if the UV254 treatment was performed under aggregating conditions (SI Figures S5 and S6). Note that the term “clumps” will be used hereafter to refer to these small aggregates formed due to the UV254 treatment, to distinguish them from the large viral aggregates formed under aggregating conditions at pH 3.0. As a result of the formation of clumps, recombination may become more important for viruses that were irradiated as aggregates compared to dispersed viruses. This is consistent with the finding that tailing occurred more readily for aggregated samples compared to dispersed ones. The occurrence of recombination furthermore explains the finding that genome degradation in the tailing part of the inactivation curve was not proportional to inactivation (Figure 1). Interestingly, given the rapid decrease in intact genomes with increasing fluence, only a negligible fraction of viruses would contain an intact polymerase gene, which is necessary to initiate the replication and recombination processes. Nevertheless, MS2 was able to replicate. This may be attributed to the ability of RNA to recombine in a nonreplicative mode, in the absence of a RNA polymerase.55,56 In the original recombination theory (also called multiplicity reactivation) Luria and Dulbecco23 assumed that a virus had n sites susceptible to inactivation which could recombine in a multiply infected cell. The probability p of a clump of j viruses to be infective after experiencing a given fluence corresponds to

only slightly larger than that of dispersed samples at pH 5.0 (blue bars, Figure 5). In contrast, if the aggregates were

Figure 5. z-average radius of MS2 determined by DLS after 1:1 dilution (and dispersion) in PBS, before (light blue) and after (red) UV254 irradiation. For each pH value two experiments were performed in 15 mM phosphate and 15 mM chloride, at an initial MS2 concentration of 4 × 1012 pfu/mL. The samples were irradiated with 219 and 389 mJ/cm2 for pH 3.0 and 5.0, respectively. Bar heights indicate the mean, and error bars indicate the lower and higher value determined for the two experiments.

exposed to UV254 before redispersion, the z-average value increased substantially. UV254-treatment of dispersed samples, however, resulted in only a negligible increase in z-average value (red bars, Figure 5). This observation indicates that some of the viruses present in an aggregate were fused during UV254 treatment, and thereafter were not fully redispersed once the pH was raised. This finding is consistent with previous reports

pj (fluence) = [1 − (1 − 10−k·fluence/ n) j ]n

(4)

Figure 6. Model fits using the modified Grant approach for four replicate inactivation experiments at pH 3.0. The model parameters corresponded to n = 7, binitial = 6.3 and bfinal = 3.6, 2.1, 3.0, and 1.3 for a, b, c, and d, respectively. 10027

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where (1 − 10−k·fluence) is the probability that a single virus is inactivated. n − 1 can be interpreted as the maximum number of times that the RNA replicase has to switch templates in order to create an intact viral genome, and k corresponds to the UV254 inactivation rate constant for dispersed viruses. Grant57 combined this theory with the clump frequency distribution proposed by Geister and Peters58 to yield the infective units (IU) upon treatment by a given fluence for solutions containing viral clumps

different levels of inactivation in each replicate experiment (Figure 1b). Despite these experimental differences, the modified Grant model was able to adequately capture the data of the four replicate experiments with a single, common value of n. An interesting feature of the model is the increase in infective virus with increasing fluence in the tailing regime (Figure 6d). This effect is evident under conditions with strong clump formation, which favors recombination. This phenomenon may contribute to the observed fluctuations in infective viruses at fluences above a 60 mJ cm−2. The model was, however, not able to describe the tailing observed at pH 5.0. This indicates that the pH 5.0 solutions were not entirely monodisperse as assumed in this work. Instead, the solutions likely contained large clumps at a very low concentration. For example, the presence of a single clump of 50 viruses in ten million dispersed viruses would explain the tailing at pH 5.0. Such a skewed clump distribution cannot be captured by the Geister and Peters expression (see eq 5).58 In summary, we argue that the tailing effect observed during UV254 treatment of viruses is neither due to light shielding nor resistant subpopulations. Instead, the presence of clumps facilitates multiple infection and recombination. Aggregation promotes the production of clumps during UV254 treatment; therefore viruses disinfected in an aggregated state exhibit more significant tailing than viruses disinfected in mostly dispersed form, even if aggregates are redispersed post-treatment. Even though the conditions used herein to induce aggregation were not environmentally relevant (high virus concentration and low pH), our findings nevertheless have implications to water treatment systems: human viruses are often released from their hosts as aggregates, and may remain aggregated in wastewater and surface water.60,61 Our results indicate that light shielding within these aggregates can be neglected. The presence of aggregates, however, can render viruses resistant to UV254, as UV254-treated aggregates are prone to recombination. For optimal disinfection results, we therefore suggest using UV254 in combination with a mild disinfectant.

∞

IU (fluence) =

∑ j−b c1 pj (fluence) j=1

(5)

where the IU is a measure of the infectivity of dispersed viruses and whole clumps in solution, c1 is the concentration of singlets, and b is a parameter reflecting the degree of clumping. This clump frequency distribution function is only defined for b −b > 1, as else Σ∞ diverges.59 j = 1j For our experimental system, the Geister and Peters frequency distribution had to be adjusted to account for the fact that clumps continuously formed during UV254 treatment, and the clump frequency distribution thus changed throughout the experiment. Hence, the b value describing the clump distribution was related to the fluence received as b = binitial − a ·fluence

(6)

where binitial reflects the degree of clumping before the UV254 treatment was initiated and a describes the change in the b value with increasing fluence. The concentration of singlets (c1; eq 5) is adjusted such that the total virus concentration remains constant with decreasing b. TEM analysis revealed an average binitial value of 6.3. The value of bfinal varied between experiments over a range of 3.2−4.4. The modified Gant approach was used to individually model four replicate experiments conducted at pH 3.0 (Figure 1b). For modeling purposes, the measured binitial of 6.3, and the k value (0.116 cm2 mJ−1) determined under nonaggregating conditions at pH 3.0 (Figure 2, red bar) were used as input parameters. Furthermore, bfinal was constrained to be >1, and the maximum clump size, jmax was set to 100, as suggested by Grant.57 Finally, the model assumes that clumps infect host cells with the same efficiency as single viruses. With a best fit of n = 7 the model was able to describe the initial sharp decay and the tailing of the experimental results with good precision (Figure 6). The n value was lower than that determined for T phages (ranging from 15 to 50 for T2, T4, T5, and T6).15 However, the T phages contain a longer genome and more genes than MS2, therefore the lower n value determined for MS2 was expected. The binitial value indicates a relatively monodisperse virus solution, consistent with the observed initial exponential decay. During UV254 irradiation permanently fused clumps started to form, resulting in a tailing due to recombination. At even higher fluences, the model predicted a further decrease in viral titer, as the additional clump formation could not compensate for the increased genome damage. Additionally, the model yielded best fit values of bfinal for each replicate experiment. The modeled bfinal spanned a slightly lower range (1.3−3.6) than the bfinal values determined by TEM (3.2−4.4). The observed range in both experimental and modeled bfinal illustrates that the clump formation due to UV254 treatment did not always occur to the same extent. This was also consistent with the fact that the onset of tailing occurred at

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ASSOCIATED CONTENT

S Supporting Information *

Descriptions of the chemicals and microorganisms used; experimental details for DLS and TEM measurements; discussion of the phosphate effect on k; k values and inactivation curves at low initial virus concentration; a model for light shielding and inactivation within viral aggregates; challenges associated with estimating aggregate size by DLS; figures showing TEM clump counts at pH 3.0 and 5.0. This information is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +41 (0)21 693 0891; fax: +41 (0)21 693 8070; e-mail: tamar.kohn@epfl.ch. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We kindly thank Peter Halter, Benoı̂t Crouzy, and Kris McNeill for the helpful discussions on light scattering and attenuation. 10028

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(20) Li, D.; Gu, A. Z.; He, M.; Shi, H.-C.; Yang, W. UV inactivation and resistance of rotavirus evaluated by integrated cell culture and realtime RT-PCR assay. Water Res. 2009, 43 (13), 3261−3269, DOI: 10.1016/j.watres.2009.03.044. (21) Galasso, G. J.; Sharp, D. G. Effect of particle aggregation on survival of irradiated Vaccinia virus. J. Bacteriol. 1965, 90 (4), 1138− 1142. (22) Sharp, D. G.; Kim, K. S. Multiplicity reactiviation and radiation survival of aggregated Vaccina virus. Calculation of plaque titer based on MR and particle aggregation seen in electron microscope. Virology 1966, 29 (3), 359−366. (23) Luria, S. E.; Dulbecco, R. Genetic recombinations leading to production of active bacteriophage from ultraviolet inactivated bacteriophage particles. Genetics 1949, 34 (2), 93−125. (24) Simon-Loriere, E.; Holmes, E. C. Why do RNA viruses recombine? Nat. Rev. Microbiol. 2011, 9 (8), 617−626, DOI: 10.1038/ nrmicro2614. (25) Nagy, P. D.; Simon, A. E. New insights into the mechanisms of RNA recombination. Virology 1997, 235 (1), 1−9, DOI: 10.1006/ viro.1997.8681. (26) Mattle, M. J.; Crouzy, B.; Brennecke, M.; Wigginton, K. R.; Perona, P.; Kohn, T. Impact of virus aggregation on inactivation by peracetic acid and implications for other disinfectants. Environ. Sci. Technol. 2011, 45 (18), 7710−7717, DOI: 10.1021/es201633s. (27) Michen, B.; Graule, T. Isoelectric points of viruses. J. Appl. Microbiol. 2010, 109 (2), 388−397, DOI: 10.1111/j.13652672.2010.04663.x. (28) Bolton, J. R.; Linden, K. G. Standardization of methods for fluence (UV dose) determination in bench-scale UV experiments. J. Environ. Eng.-ASCE 2003, 129 (3), 209−215, DOI: 10.1061/(asce) 0733-9372(2003)129:3(209). (29) Rahn, R. O. Potassium iodide as a chemical actinometer for 254 nm radiation: Use of iodate as an electron scavenger. Photochem. Photobiol. 1997, 66 (4), 450−455. (30) Pecson, B. M.; Ackermann, M.; Kohn, T. Framework for using quantitative PCR as a nonculture based method to estimate virus infectivity. Environ. Sci. Technol. 2011, 45 (6), 2257−2263, DOI: 10.1021/es103488e. (31) Schwarzenbach, R. P.; Gschwend, P. M.; Imboden, D. M. Environmental Organic Chemistry; Wiley: Hoboken, NJ, 2003. (32) Morowitz, H. J. Absorption effects in volume irradiation of microorganisms. Science 1950, 111 (2879), 229−230. (33) Voet, D.; Gratzer, W. B.; Cox, R. A.; Doty, P. Absorption spectra of nucleotides, polynucleotides, and nucleic acids in the far ultraviolet. Biopolymers 1963, 1 (3), 193−208, DOI: 10.1002/bip.360010302. (34) Sommer, R.; Haider, T.; Cabaj, A.; Pribil, W.; Lhotsky, M. Time dose reciprocity in UV disinfection of water. Water Sci. Technol. 1998, 38 (12), 145−150, DOI: 10.1016/s0273-1223(98)00816-6. (35) Shin, G.-A.; Lee, J.-K.; Linden, K. G. Enhanced effectiveness of medium-pressure ultraviolet lamps on human adenovirus 2 and its possible mechanism. Water Sci. Technol. 2009, 60 (4), 851−857, DOI: 10.2166/wst.2009.414. (36) Acharya, P.; Cheruku, P.; Chatterjee, S.; Acharya, S.; Chattopadhyaya, J. Measurement of nucleobase pK(a) values in model mononucleotides shows RNA-RNA duplexes to be more stable than DNA-DNA duplexes. J. Am. Chem. Soc. 2004, 126 (9), 2862− 2869, DOI: 10.1021/ja0384546. (37) Dika, C.; Duval, J. F. L.; Ly-Chatain, H. M.; Merlin, C.; Gantzer, C. Impact of internal RNA on aggregation and electrokinetics of viruses: Comparison between MS2 phage and corresponding virus-like particles. Appl. Environ. Microbiol. 2011, 77 (14), 4939−4948, DOI: 10.1128/aem.00407-11. (38) ViralZone of the Swiss Institue of Bioinformatics Website; http://viralzone.expasy.org/. (39) Gallagher, S. R. Quantitation of DNA and RNA with absorption and fluorescence spectroscopy. In Current Protocols in Molecular Biology; John Wiley & Sons, Inc., 2011; Vol. Supplement 93.10.1002/ 0471142727.mba03ds93.

This study was funded by the Swiss National Science Foundation (projects 118077 and 131918).

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REFERENCES

(1) American Water Works Association. Waterborne Pathogens; American Water Works Association: Denver, CO, 2006. (2) Galbraith, N. S.; Barrett, N. J.; Stanwellsmith, R. Water amd disease after Croydon - A review of water-borne and water-associated disease in the UK 1937−86. J. Inst. Water Environ. Manage. 1987, 1 (1), 7−21. (3) Gerardi, M. H.; Zimmerman, M. C. Wastewater Pathogens; WileyInterscience: Hoboken, NJ, 2005. (4) Kruithof, J. C.; Van der Leer, R. C.; Hijnen, W. A. M. Practical experiences with UV disinfection in the Netherlands. J. Water SRT Aqua 1992, 41 (2), 88−94. (5) Clancy, J. L.; Hargy, T. M.; Marshall, M. M.; Dyksen, J. E. UV light inactivation of Cryptosporidium oocysts. J. Am. Water Works Assoc. 1998, 90 (9), 92−102. (6) Shin, G. A.; Linden, K. G.; Arrowood, M. J.; Sobsey, M. D. Lowpressure UV inactivation and DNA repair potential of Cryptosporidium parvum oocysts. Appl. Environ. Microbiol. 2001, 67 (7), 3029−3032, DOI: 10.1128/aem.67.7.3029-3032.2001. (7) Craik, S. A.; Finch, G. R.; Bolton, J. R.; Belosevic, M. Inactivation of Giardia muris cysts using medium-pressure ultraviolet radiation in filtered drinking water. Water Res. 2000, 34 (18), 4325−4332, DOI: 10.1016/s0043-1354(00)00207-4. (8) Sedlak, D. L.; von Gunten, U. The chlorine dilemma. Science 2011, 331 (6013), 42−43, DOI: 10.1126/science.1196397. (9) Bellar, T. A.; Lichtenberg, J. J.; Kroner, R. C. The occurence of organohalides in chlorinated drinking waters. J. Am. Water Works Assoc. 1974, 66 (12), 703−706. (10) Chang, J. C. H.; Ossoff, S. F.; Lobe, D. C.; Dorfman, M. H.; Dumais, C. M.; Qualls, R. G.; Johnson, J. D. UV inactivation of pathogenic and indicator microorganisms. Appl. Environ. Microbiol. 1985, 49 (6), 1361−1365. (11) Sommer, R.; Weber, G.; Cabaj, A.; Wekerle, J.; Keck, G.; Schauberger, G. UV-inactivation of microorganisms in water. Zentralbl. Hyg. Umweltmed. 1989, 189 (3), 214−224. (12) Hijnen, W. A. M.; Beerendonk, E. F.; Medema, G. J. Inactivation credit of UV radiation for viruses, bacteria and protozoan (oo)cysts in water: A review. Water Res. 2006, 40 (1), 3−22, DOI: 10.1016/ j.watres.2005.10.030. (13) Rainbow, A. J.; Mak, S. DNA damage and biological function of human Adenovirus after UV-irradiation. Int. J. Radiat. Biol. 1973, 24 (1), 59−72, DOI: 10.1080/09553007314550821. (14) Nieuwstad, T. J.; Havelaar, A. H. The kinetics of batch ultraviolet inactivation of bacteriophage-MS2 and microbiological calibration of an ultraviolet pilot-plant. J. Environ. Sci. Health A 1994, 29 (9), 1993−2007. (15) Luria, S. E. Reactivation of irradiated bacteriophage by transfer of self-reproducing units. Proc. Natl. Acad. Sci., U.S.A. 1947, 33 (9), 253−264, DOI: 10.1073/pnas.33.9.253. (16) Battigelli, D. A.; Sobsey, M. D.; Lobe, D. C. The Inactivation of Hepatitis A Virus and Other Model Viruses by UV Irradiation. Water Sci. Technol. 1993, 27 (3−4), 339−342. (17) Cutler, T.; Wang, C.; Qin, Q.; Zhou, F.; Warren, K.; Yoon, K. J.; Hoff, S. J.; Ridpath, J.; Zimmerman, J. Kinetics of UV(254) inactivation of selected viral pathogens in a static system. J. Appl. Microbiol. 2011, 111 (2), 389−395, DOI: 10.1111/j.13652672.2011.05046.x. (18) Thurston-Enriquez, J. A.; Haas, C. N.; Jacangelo, J.; Riley, K.; Gerba, C. P. Inactivation of feline calicivirus and adenovirus type 40 by UV radiation. Appl. Environ. Microbiol. 2003, 69 (1), 577−582, DOI: 10.1128/aem.69.1.577-582.2003. (19) Simonet, J.; Gantzer, C. Inactivation of poliovirus 1 and Fspecific RNA phages and degradation of their genomes by UV irradiation at 254 nm. Appl. Environ. Microbiol. 2006, 72 (12), 7671− 7677, DOI: 10.1128/aem.01106-06. 10029

dx.doi.org/10.1021/es302058v | Environ. Sci. Technol. 2012, 46, 10022−10030

Environmental Science & Technology

Article

(61) Young, D. C.; Sharp, D. G. Poliovirus aggregates and their survival in water. Appl. Environ. Microbiol. 1977, 33 (1), 168−177.

(40) Loge, F. J.; Emerick, R. W.; Thompson, D. E.; Nelson, D. C.; Darby, J. L. Factors influencing ultraviolet disinfection performance part I: Light penetration to wastewater particles. Water Environ. Res. 1999, 71 (3), 377−381, DOI: 10.2175/106143097x122176. (41) Templeton, M. R.; Andrews, R. C.; Hofmann, R. Inactivation of particle-associated viral surrogates by ultraviolet light. Water Res. 2005, 39 (15), 3487−3500, DOI: 10.1016/j.watres.2005.06.010. (42) Das, G.; Sil, K.; Das, J. Repair of Ultraviolet-Light-Induced DNA Damage in Vibrio-Cholerae. Biochim. Biophys. Acta 1981, 655 (3), 413−420, DOI: 10.1016/0005-2787(81)90053-8. (43) Harris, G. D.; Adams, V. D.; Sorensen, D. L.; Curtis, M. S. Ultraviolet inactivation of selected bacteria and viruses with photoreactivation of the bacteria. Water Res. 1987, 21 (6), 687−692, DOI: 10.1016/0043-1354(87)90080-7. (44) Sommer, R.; Lhotsky, M.; Haider, T.; Cabaj, A. UV inactivation, liquid-holding recovery, and photoreactivation of Escherichia coli O157 and other pathogenic Escherichia coli strains in water. J. Food Protect. 2000, 63 (8), 1015−1020. (45) Barr, J.; Fearns, R. How RNA viruses maintain their genome integrity. J. Gen. Virol. 2010, 91, 1373−1387, DOI: 10.1099/ vir.0.020818-0. (46) Lin, P. F.; Howardflanders, P. Genetic exchanges caused by ultraviolet photoproducts in phage-lambda DNA-molecules - role of DNA-replication. Mol. Gen. Genet. 1976, 146 (2), 107−115, DOI: 10.1007/bf00268079. (47) Hirst, G. K. Genetic recombination with Newcastle disease virus, Poliovirus, and Influenza. Cold Spring Harb. Symp. 1962, 27, 303−&. (48) Ledinko, N. Genetic recombination with Poliovirus type 1 studies of crosses between a normal horse serum-resistant mutant and several guanidine-resistant mutants of the same strain. Virology 1963, 20 (1), 107−&, DOI: 10.1016/0042-6822(63)90145-4. (49) Lai, M. M. C. RNA recombination in animal and plant-viruses. Microbiol. Rev. 1992, 56 (1), 61−79. (50) Olsthoorn, R. C. L.; vanDuin, J. Evolutionary reconstruction of a hairpin deleted from the genome of an RNA virus. Proc. Natl. Acad. Sci. U.S.A. 1996, 93 (22), 12256−12261, DOI: 10.1073/pnas.93.22.12256. (51) Abel, P. Multiplicity reactivation and marker rescue with Vaccinia virus. Virology 1962, 17 (4), 511−519, DOI: 10.1016/00426822(62)90150-2. (52) Young, D. C.; Sharp, D. G. Partial reactivation of chlorinetreated echovirus. Appl. Environ. Microbiol. 1979, 37 (4), 766−773. (53) Pattison, D. I.; Rahmanto, A. S.; Davies, M. J. Photo-oxidation of proteins. Photochem. Photobiol. Sci. 2012, 11 (1), 38−53, DOI: 10.1039/c1pp05164d. (54) Michnik, A.; Michalik, K.; Drzazga, Z. Effect of UVC radiation on conformational restructuring of human serum albumin. J. Photochem. Photobiol. B-Biol. 2008, 90 (3), 170−178, DOI: 10.1016/ j.jphotobiol.2007.12.007. (55) Gmyl, A. P.; Belousov, E. V.; Maslova, S. V.; Khitrina, E. V.; Chetverin, A. B.; Agol, V. I.; Nonreplicative, R. N. A. recombination in poliovirus. J. Virol. 1999, 73 (11), 8958−8965. (56) Gmyl, A. P.; Korshenko, S. A.; Belousov, E. V.; Khitrina, E. V.; Agol, V. I. Nonreplicative homologous RNA recombination: Promiscuous joining of RNA pieces? RNA 2003, 9 (10), 1221− 1231, DOI: 10.1261/rna.5111803. (57) Grant, S. B. Inactivation kinetics of viral aggregates. J. Environ. Eng.-ASCE 1995, 121 (4), 311−319. (58) Geister, R. G.; Peters, D. H. A. Quantitative use of electron microscope in virus research - Method of analyzing and predicting biologic titers of aggregated virus suspensions with a new law of aggregation. Lab. Invest. 1965, 14 (6P2), 864−874. (59) Abramowitz, M.; Stegun, I. A. Handbook of Mathematical Functions: With Formulas, Graphs and Mathematical Tables; Wiley: New York, 1972. (60) Narang, H. K.; Codd, A. A. Frequency of pre-clumped virus in routine fecal specimens from patients with acute non-bacterial gastroenteritis. J. Clin. Microbiol. 1981, 13 (5), 982−988. 10030

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