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Wavelength-Dependent Damage to Adenoviral Proteins Across the Germicidal UV Spectrum Sara E. Beck,† Natalie M. Hull, Christopher Poepping, and Karl G. Linden* Department of Civil, Environmental, and Architectural Engineering, University of Colorado Boulder, UCB 428, Boulder, Colorado 80309, United States S Supporting Information *

ABSTRACT: Adenovirus, a waterborne pathogen responsible for causing bronchitis, pneumonia, and gastrointestinal infections, is highly resistant to UV disinfection and therefore drives the virus disinfection regulations set by the U.S. Environmental Protection Agency. Polychromatic UV irradiation has been shown to be more effective at inactivating adenovirus and other viruses than traditional monochromatic irradiation emitted at 254 nm; the enhanced efficacy has been attributed to UV-induced damage to viral proteins. This research shows UV-induced damage to adenoviral proteins across the germicidal UV spectrum at wavelength intervals between 200 and 300 nm. A deuterium lamp with bandpass filters and UV light-emitting diodes (UV LEDs) isolated wavelengths in approximate 10 nm intervals. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and image densitometry were used to detect signatures for the hexon, penton, fiber, minor capsid, and core proteins. The greatest loss of protein signature, indicating damage to viral proteins, occurred below 240 nm. Hexon and penton proteins exposed to a dose of 28 mJ/cm2 emitted at 214 nm were approximately 4 times as sensitive and fiber proteins approximately 3 times as sensitive as those exposed to a dose of 50 mJ/cm2 emitted at 254 nm. At 220 nm, a dose of 38 mJ/cm2 reduced the hexon and penton protein quantities to approximately 33% and 31% of the original amounts, respectively. In contrast, a much higher dose of 400 mJ/cm2 emitted at 261 and 278 nm reduced the original protein quantity to between 66−89% and 80−93%, respectively. No significant damage was seen with a dose of 400 mJ/cm2 at 254 nm. This research directly correlates enhanced inactivation at low wavelengths with adenoviral protein damage at those wavelengths, adding fundamental insight into the mechanisms of inactivation of polychromatic germicidal UV irradiation for improving UV water disinfection.

1. INTRODUCTION Adenovirus is a common cause of respiratory illnesses, including bronchitis and pneumonia, as well as gastrointestinal infections. Immunocompromised individuals, infants, and children are the most at risk of adenovirus infection; however, it has historically caused acute respiratory illness in military recruits as well.1 As a waterborne pathogen, adenovirus has been found in both contaminated and treated drinking water2,3 and raw and treated wastewaters.4 It is of significant interest in the water treatment industry, particularly with regard to ultraviolet (UV) disinfection, because of its greater resistance to UV irradiation relative to other pathogens. In disinfection with traditional low-pressure (LP) UV irradiation emitting at 254 nm, 4-log inactivation of adenovirus type 2 occurs at a UV dose approximately 4 times greater than that required to inactivate other enteric viruses, including echovirus, coxsackievirus, and poliovirus.5−8 This increased resistance drives regulations set by the U.S. Environmental Protection Agency (EPA) for virus inactivation by UV irradiation. To demonstrate 4-log inactivation of all viruses, for example, the Surface Water Treatment Rule requires a UV dose of 186 mJ/cm2 on the basis of empirical results on adenovirus inactivation by LP UV © XXXX American Chemical Society

irradiation at 254 nm; this dose is 4−7 times greater than standard UV doses applied for water disinfection (25−40 mJ/ cm2).9,10 Despite its demonstrated resistance to monochromatic 254 nm irradiation emitted from LP UV lamps, adenovirus has shown an increased sensitivity to polychromatic UV irradiation from medium-pressure (MP) mercury vapor lamps emitting across the germicidal UV spectrum. MP UV lamps were 2−4 times more effective than LP UV lamps,8,11−14 inactivating adenovirus by 4-log at doses of 40−80 mJ/cm2. This enhanced viral inactivation from polychromatic lamps has been attributed, in large part, to UV-induced damage to viral proteins. When damage to adenoviral DNA was measured with a polymerase chain reaction (PCR) assay, the monochromatic and polychromatic light sources were equally effective, inducing the same amount of DNA lesions per kilobase.15 The enhanced susceptibility of adenovirus 2 to polychromatic UV irradiation Received: Revised: Accepted: Published: A

September 7, 2017 November 12, 2017 November 27, 2017 November 27, 2017 DOI: 10.1021/acs.est.7b04602 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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Environmental Science & Technology

CO2. They were infected with stock HAdV2 and incubated for up to 2 weeks or until cytopathogenic effects (CPE) were observed. Cells underwent 3 freeze−thaw cycles, and a portion of the cell lysate was passaged onto fresh A549 cells for further propagation. Cell lysates from all passages were combined to obtain maximal viral stock. Lysates were centrifuged at 2500g for 30 min to remove cell debris. The supernatant containing virus was then centrifuged at 10000g for 10 min to remove any remaining small cellular debris. In order to remove any potential viral clumps, remove any remnant of the growth medium, and further purify the viral stock, the virus supernatant was further concentrated using celite as described by McMinn et al.27 Following celite concentration, viral stocks were further concentrated using 30 kDa molecular weight cutoff Vivaspin-20 ultrafilters (Sartorius-Stedim, Aubagne, France) as described previously.28 Viral stocks, estimated by a quantal assay29 at 3.2 × 109 MPN/mL, were shipped to the University of Colorado Boulder and stored at −80 °C until UV exposure experiments. 2.2. UV Irradiations. HAdV2 suspensions, diluted 50% with sterile filtered water (Sigma-Aldrich, St Louis, MO), were irradiated by a deuterium lamp, UV LEDs, and an LP mercury vapor lamp. The spectrum obtained using the 200 W deuterium lamp (Hi-Tech Lamps, Mountain View, CA) was attenuated with bandpass filters to filter its emissions to roughly 10 nm intervals within the germicidal UV-C range. The filtered deuterium lamp spectra (Figure 1), measured with a Maya

was therefore due to the sensitivity of viral components other than nucleic acid.16 Since adenovirus comprises only nucleic acid and proteins, the increased susceptibility was due to damage to viral proteins.17 Adenoviral proteins play an integral role in the infection process. Fiber proteins protruding from 12 vertices initiate contact with the host cell receptor, primarily the cocksackie− adenovirus receptor (CAR) on human lung cells.18 The penton base of the fiber protein is responsible for entry of the virion into the host cell; it binds to integrin coreceptors on the cell surface, which stimulates endocytosis and results in internalization of the virus.19,20 The hexon protein, characterized for its stability due to a highly folded design, maintains the structural stability of the virus during infection.18,21 Minor capsid polypeptide IIIa binds to the penton, hexon, and other capsid proteins. Polypeptides VI and VIII, which bridge the DNA core to the viral capsid, enable the entry of the viral DNA, core protein V, and the terminal protein into the host cell’s nucleus.18,22 UV-induced damage to these viral proteins could therefore prevent the virus from attaching to and entering a host cell, from maintaining its structural stability during infection, and from releasing DNA into the cell nucleus to begin replication. Although UV inactivation of adenovirus was most pronounced at wavelengths below 240 nm,16 past studies had not directly correlated that enhanced inactivation with adenoviral protein damage at those UV wavelengths. The present study measures the protein damage that occurs at specific wavelength intervals between 200 and 300 nm. A deuterium lamp with bandpass filters and UV light-emitting diodes (UV LEDs) were used to isolate UV wavelengths in approximate 10 nm intervals. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate the proteins by molecular weight; the hexon, penton, minor capsid, fiber, and core protein bands were analyzed for UV-induced damage. Understanding the fundamental mechanisms of virus inactivation has significant applications for disinfection with novel sources of polychromatic UV light, including UV LEDs. Deep-UV LEDs, which emit in the UV-C range, are gaining popularity because of their absence of mercury, low power requirements, ability to be turned on and off instantaneously with no required warmup, and small size, which allows for unique reactor designs.23−25 The opportunity to select UV LEDs with specific wavelength emissions for tailored UV disinfection makes this research especially relevant.26 UV LEDs have been tested for inactivation of adenovirus in the DNA absorbance region between 260 and 285 nm.24,26 This work gives additional insight into the virus inactivation mechanisms of UV LEDs emitting in the low-wavelength region, below 240 nm.

Figure 1. Normalized relative emission spectra from the deuterium lamp with bandpass filters, the UV LEDs, and the LP UV mercury vapor lamp.

2000 Pro spectrometer (Ocean Optics, Dunedin, FL), exhibited peak wavelength emissions at 213.4, 220.0, 226.3, 239.8, and 267.6 nm with full width at half-maximum (fwhm) bandwidths measuring between 9.6 and 12.4 nm. The spectra from the turnkey UV-C LED unit (Dot Metrics Technologies, Charlotte, NC), measured with the Maya 2000 Pro spectrometer, peaked at 259.6 and 276.6 nm with fwhm bandwidths of 12.6 and 9.8 nm, respectively (Figure 1). The deuterium lamp and UV LED sources were set up in a bench-scale collimated beam apparatus as described by Bolton and Linden.30 For reference, HAdV2 suspensions were also irradiated with a custom-made LP UV system built with four 15 W mercury vapor lamps emitting monochromatic irradiation at 253.7 nm (Figure 1) through a 4 in. aperture. The HAdV2 exposures were conducted across the germicidal UV spectrum. Specific wavelengths were analyzed in more detail. The wavelength of 220 nm was of particular interest, as protein damage is expected to be greatest at wavelengths below 240 nm because of the high protein absorbance.16,31,32 At 220 nm, the virus was exposed to UV doses of 7.6, 15.1, 22.7, 30.3, and 37.8 mJ/cm2. At the common reference, the LP UV

2. MATERIALS AND METHODS 2.1. Adenovirus Propagation and Enumeration. Adenovirus stocks, provided by the EPA in Cincinnati, OH, were prepared as follows: Human adenovirus 2 (HAdV2) (ATCC VR-846; American Type Tissue Collection, Manassas, VA) was propagated in A549 human lung carcinoma cells (ATCC CCL-185). The cells were planted in 175 cm2 vented tissue culture flasks (Greiner, Monroe, NC) in Dulbecco’s minimum essential medium (DMEM) (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% fetal calf serum (Thermo Fisher Scientific) and maintained in 5% (v/v) B

DOI: 10.1021/acs.est.7b04602 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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spiked with 2 μL of 0.5 mg/mL aprotinin, an internal protein standard (MP Biomedicals, Santa Ana, CA), and pretreated by incubation for 10 min at room temperature with 32 μL of 2% sodium deoxycholate (Sigma-Aldrich). Samples were precipitated with 10% trichloroacetic acid, adding 25% by volume (37.5 μL), and chilled on ice for 30 min. Samples were centrifuged for 30 min at 4 °C (20000g) to pelletize the protein. The acidic supernatant was decanted, and the protein pellet was washed with acetone (800 μL, −20 °C) and centrifuged again. The precipitated and dried protein pellets were reduced and denatured in a solution of Laemmli buffer (Bio-Rad, Berkeley, CA) with 5% β-mercaptoethanol (15 μL) and SDS electrophoresis running buffer (9 μL, 25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3) (Bio-Rad). Resuspended proteins were heated to 95 °C to further denature the proteins prior to injection into 4−20% gradient Tris-HCl precast gels (Bio-Rad). After gel electrophoresis (150 V, 95 min), the gels were fixed in a 7% acetic acid/10% methanol solution for 30 min with gentle agitation, stained overnight with SYPRO Ruby protein gel stain (Bio-Rad) according to manufacturer’s instructions, and destained with the acetic acid methanol solution. Gels were imaged with a Gel-Doc imager (Bio-Rad), and individual protein bands were identified by molecular weight18,35−37 with the aid of the broad-range molecular weight protein standard (Bio-Rad). Proteins were quantified from image densitometry using GelAnalyzer software (www. gelanalyzer.com) relative to the internal protein standard (aprotinin, 6.5 kDa). Data from the duplicate SDS PAGE gels were averaged; error bars represent ±1 standard deviation of the mean.

wavelength of 254 nm, protein damage was measured at UV doses of 100, 200, 300, and 400 mJ/cm2. Protein damage from both LEDs, emitting at weighted average wavelengths of 261 and 278 nm, was also measured at UV doses of 100, 200, 300, and 400 mJ/cm2; protein damage at 278 nm was of research importance because of its proximity to a relative peak of protein absorbance.32 HAdV2 suspensions were exposed to the UV sources in continuously stirred 2 mL volumes (3.5 cm diameter Petri dishes, 0.2 cm sample depth). Irradiance was measured at the water surface with an IL-1700 radiometer, SED 240 detector, and W-diffuser (International Light, Peabody, MA). For exposures involving the deuterium lamp with bandpass filters, the incident irradiance was lower than for the other two sources, ranging from 0.0102 to 0.025 mW/cm2. For the UV LEDs, the irradiance ranged from 0.193 to 0.337 mW/cm2. For the LP UV lamp, the irradiance at the water surface was 0.95 mW/cm2. Adenovirus is expected to follow the rule of time dose reciprocity, as do the bacterial viruses MS2, phix174, and B40-8, meaning that the microbicidal effect of the UV irradiation is due to the UV dose applied over the range of exposure times.33 The incident irradiance values and exposure times are given in Table S1 in the Supporting Information. Average UV doses for the collimated beam tests were determined as described previously,26 adjusting for reflection off the water surface, UV absorbance across each wavelength range (Cary 100 spectrophotometer, Agilent Technologies, Santa Clara, CA), and depth of the water sample as well as the nonuniformity of the distribution of light across the surface of the sample. Petri factors ranged from 0.90 to 0.92 for the deuterium lamp with bandpass filters, 0.88 to 0.93 for the UV LEDs, and 1.0 for the LP UV mercury vapor lamp. Since the deuterium lamp with bandpass filters and the UV LEDs are polychromatic light sources, the UV dose accounted for the relative lamp emission (RLE) of the light source (Figure 1) and the sensitivity of the radiometer used to measure the irradiance, which was given with the radiometer calibration data.10,34 The RLE and radiometer sensitivity were taken relative to the weighted average wavelength of each source (i.e., 214, 220, 227, 240, and 268 nm for the deuterium lamp and 261 and 278 nm for the UV LEDs). Average irradiance was used for the UV dose calculations; they were not weighted for the DNA absorbance spectrum or the action spectrum. Irradiations were conducted at room temperature. The deuterium lamp operated on a chiller, which cooled the lamp unit to between 2 and 15 °C. The LED unit contained a closedloop thermal management system to regulate internal UV LED temperatures. Sample volumes were measured after irradiation. For the deuterium lamp exposures, from 0% to 10% of the volume, and therefore the sample depth, was lost to evaporation. For the UV LED exposures, from 0% to 20% of the volume was lost. In the case of evaporation, purified water was added back to each sample before analysis. The volume of water lost and added back for each sample is given in Table S1. Samples were placed in a −80 °C freezer prior to the SDS PAGE assay. Two independent UV irradiation experiments were conducted for each UV dose. Duplicate protein precipitations and SDS-PAGE gels were analyzed for each independent exposure. 2.3. SDS-PAGE Analysis. HAdV2 protein signatures were measured by SDS-PAGE using a method modified from that described previously.15 Of the 2 mL irradiated samples, 150 μL of each sample was used for protein analysis. Each volume was

3. RESULTS AND DISCUSSION The SDS-PAGE gel in Figure 2 shows the adenoviral protein signature across the germicidal UV spectrum. Proteins were

Figure 2. SDS-PAGE gel depicting HAdV2 protein sensitivity across the germicidal UV spectrum.

identified by the molecular weight (kDa) labeled on the left side of the image, which corresponds to the protein ladder in lane 1. Lanes 2−8 show adenoviral proteins across the UV spectrum after exposure to the wavelengths and doses given below the image. The UV dose was 50 mJ/cm2 at wavelengths of 240 nm and above. Below 240 nm, the UV doses steadily decreased to ensure protein detection by the SDS-PAGE assay. Adenoviral proteins, labeled on the right-hand side, were historically assigned Roman numerals corresponding to their migration order through the gel, in decreasing molecular weight order.38 In this present study, the hexon (polypeptide II, 109 kDa), penton base (polypeptide III, 63.3 kDa), minor capsid C

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Figure 3. Sensitivities of HAdV2 proteins across the germicidal UV spectrum relative to their sensitivities at 254 nm in comparison to the HAdV2 action spectrum (inset).16 The lower UV doses applied at 214, 220, and 227 nm should be noted. Error bars represent 1 standard deviation of the average of two gels for two independent data sets.

Figure 4. SDS-PAGE gels for adenoviral proteins following exposure to increasing doses of UV light emitted at 220, 254, 261, and 278 nm. The differences in UV doses used for 220 nm should be noted.

proteins approximately 4 times as much and the fiber proteins approximately 3 times as much as a dose of 50 mJ/cm2 emitted at 254 nm. This work is consistent with previous work that showed increased viral capsid damage to adenovirus 5 at the lower UV wavelengths measured, approximately 230−245 nm, than at wavelengths above 245 nm.38 At wavelengths below 240 nm, the UV absorbance of proteins is highest, primarily because of the high absorbance of the peptide bond.31,32 Peptide bonds are prevalent in protein structures as the links between the amino acids forming the protein structure. Increased protein damage from 214 nm irradiation was also evident, though less pronounced, in core proteins V and VII. Eischeid et al.15 also reported that the core proteins were less susceptible to UV damage, perhaps because they were shielded from the UV irradiation by the viral DNA molecule, which is wound around the DNA−protein complex.40 The response of adenoviral proteins to increasing doses of UV light emitted at 220, 254, 261, and 278 nm is shown in the SDS-PAGE images in Figure 4. In the image for the 220 nm exposures, the protein signature is strong in the unexposed (0 mJ/cm2) sample, whereas the exposed samples are progressively degraded. Individual protein bands are less distinguishable because of protein aggregation;42 however, a strong signature is maintained for the aprotinin protein standard. A higher protein signature is also seen in the injection wells, an indication of UV-induced aggregation.42 Gels

(IIIa, 65.3 kDa or 63.3 kDa), fiber (IV, 62 kDa) and core (V, 41.7 kDa, and VII, 22 kDa) proteins were selected for analysis.18,35,37,39 It should be noted that the UniProt Database lists protein IIIa with a molecular weight of 65.3 kDa, placing it above the penton protein (III) in the SDS PAGE gel. This research follows other references, which list a lower molecular weight or show the protein order reversed as indicated in Figure 2.35,37,40,41 The gel indicates a decreasing adenovirus protein signature with decreasing wavelength below 240 nm. The spiked aprotinin protein standard (6.5 kDa) is strong across all lanes of the gel. In the 214, 220, and 227 nm lanes, the protein signature of the virus is very weak. Individual protein bands are less distinguishable and are blended throughout each lane, which is an indication of UV-induced protein aggregation.42 The low-wavelength lanes also show the strongest protein signature in the injection well, at the top of the image; this is further indication of the aggregation of proteins into a macromolecule, which cannot be reduced and denatured for migration through the gel.42 Image densitometry analysis of the protein gel in Figure 2 yielded the quantitative relative protein sensitivity data shown in Figure 3. The relative protein sensitivity followed a similar trend as the UV action spectra of adenovirus 2 (Figure 3 inset,16), increasing prominently below 240 nm. A UV dose of 28 mJ/cm2 emitted at 214 nm damaged the hexon and penton D

DOI: 10.1021/acs.est.7b04602 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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Figure 5. Quantities of adenoviral protein after exposure to increasing doses of UV light at 220 nm.

Figure 6. Quantities of adenoviral protein after exposure to UV light emitted at 254, 261, and 278 nm.

for protein signatures after exposure to UV wavelengths of 254, 261, and 278 nm (Figure 4) showed much less protein degradation as well as less protein aggregation in the lanes and injection wells. The damage induced by 220 nm UV light is quantified in Figure 5, where it can be seen that a 38 mJ/cm2 dose of 220 nm irradiation reduced the hexon and penton protein quantities to approximately 33% and 31% of the original amounts, respectively. UV-induced damage was less pronounced in the minor capsid, fiber, and core proteins, with 47%−54% of the original protein content detected. In contrast, the protein content in the injection well, shown in Figures 4 and 5, tended to increase with increasing dose, reflecting increasing UVinduced protein aggregation. As mentioned above, the UV absorbance of proteins is high in this low-wavelength range, primarily because of the absorbance of the peptide bonds.31,32

Aromatic amino acids, including tryptophan, tyrosine, and phenylalanine also strongly absorb UV irradiation at 220 nm.31 In contrast to the protein damage induced at 220 nm with low UV doses of up to 37.8 mJ/cm2, significantly less protein damage was seen at 254, 261, and 278 nm at higher doses of up to 400 mJ/cm2 . Image densitometry quantification of adenoviral protein damage from 254, 261, and 278 nm UV irradiation is given in Figure 6. For UV irradiation emitted at 254 nm by the low-pressure mercury vapor lamp, little to no protein degradation was detected in the gel at doses as high as 400 mJ/cm2. These results are contrary to a previous study also using HAdV2, which showed some (up to 40%) degradation of various proteins following LP UV irradiation at a dose of 300 mJ/cm2.17 For UV irradiation emitted by a UV-C LED at 261 nm, image densitometry quantification detected 66−89% of the original protein content following the highest UV dose of 400 E

DOI: 10.1021/acs.est.7b04602 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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Environmental Science & Technology mJ/cm2 (Figure 6). For UV-C LED irradiation at 278 nm at the same high dose of 400 mJ/cm 2 , image densitometry quantification showed 80−93% of the original protein content. In general, proteins have a relative peak UV absorbance near 280 nm due to the absorbance of the tryptophan and tyrosine aromatic amino acids as well as the cystine disulfide bond.31,44 One method for determining protein quantity involves measuring the UV absorbance of a compound at 280 nm.45 Therefore, more protein degradation was expected near 280 nm; however, UV irradiation emitted by the UV-C LED at 278 nm did not appear to induce significant protein damage compared with 254 or 261 nm up to a UV dose of 400 mJ/cm2. This agrees with recent work assessing adenovirus infectivity near these wavelengths. In a study of adenovirus inactivation measured by cell culture, the viral inactivation rates at 253.7, 260, and 280 nm were very similar (0.030, 0.029, and 0.032 cm2/mJ, respectively).16 At these wavelengths, adenovirus inactivation is dominated by DNA damage,16 in part because the absorbance of amino acids is an order of magnitude lower than the DNA absorbance.32 The SDS-PAGE assay was sufficient to indicate protein damage to the adenovirus 2 hexon, penton, minor capsid, fiber, and core proteins, particularly at wavelengths below 240 nm. This research supplements previous work16,17 by showing protein damage at low UV wavelengths below 240 nm, which is responsible for the enhanced efficacy of MP UV inactivation of adenovirus compared to LP UV inactivation. Future research would benefit by analyzing the response of adenoviral proteins further at 270 nm. The UV inactivation rate of adenovirus, measured with cell culture infectivity, was higher at 270 nm than at 260 or 280 nm.16 This could be due in part to the fact that the relative peak UV absorbance of intact, purified adenovirus 2 (measured in this study, not shown) is 270 nm. Unfortunately, for this work, additional protein analysis with irradiation at 268 nm, which required a deuterium lamp with bandpass filters, was not feasible because of the long exposure times (>14 h) required for doses up to 400 mJ/cm2; these long exposures would have resulted in experimental complications and potential errors. Future research could expand on this work by utilizing more advanced protein detection methods that have recently been employed in the field, including ethidium monoazide treatment polymerase chain reaction (EMA-PCR), matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry, and stable isotope labeling of amino acids in cell culture (SILAC). EMA-PCR was used to measure the viral capsid integrity of adenovirus 5 following exposure to UV light.43 MALDI-TOF mass spectrometry has been used to demonstrate UV-induced damage to viral capsid proteins.46 SILAC has been used to localize the UV-induced protein damage specifically with respect to human adenovirus.47 All three methods could be employed to further quantify UVinduced protein damage across the germicidal UV spectrum and corroborate the results of this study. This work directly correlates enhanced inactivation of adenovirus at low UV wavelengths (below 240 nm) with adenoviral protein damage at those wavelengths, adding fundamental insight into the mechanisms of inactivation of polychromatic germicidal UV irradiation for improving UV water disinfection. Protein damage at 220 nm was significantly greater than at 254, 261, or 278 nm. These results provide further evidence that polychromatic UV light, such as that emitted by medium-pressure UV sources used in practice,

inactivates some viruses differently than bacteria by damaging viral proteins and impacting the virus’s ability to infect. Thus, it complements previous studies that evaluated low-wavelength emissions and pathogen response with the goal of ensuring that UV disinfection systems are adequately protecting human health. The results also inform future applications where selection of wavelengths using UV LEDs or other novel wavelength-specific UV sources may be possible in a tailoredemission UV disinfection system.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.7b04602. Details of experiments conducted with the deuterium, low-pressure UV, and UV LED light sources (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; phone: 303-492-4798; fax: 303-492-7317. ORCID

Natalie M. Hull: 0000-0003-2876-6721 Karl G. Linden: 0000-0003-4301-7227 Present Address †

S.E.B.: Department of Environmental Microbiology, Eawag: Swiss Federal Institute of Aquatic Science and Technology, 8600 Dubendorf, Switzerland. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge Ms. Jennifer Cashdollar from the U.S. EPA for providing purified HAdV2 stock at high concentration as well as her technical expertise on adenovirus quantification and propagation. We thank Dr. Hodon Ryu from the EPA for his technical expertise and assistance with reviewer comments. We thank Dr. Tamar Kohn for assistance with viral protein identification. This research was partially funded by EPA Science to Achieve Results (STAR) Fellowship Assistance Agreement FP91709801 for S.E.B. and Grant 83560301 for the Design of Risk-reducing, Innovative-implementable Smallsystem Knowledge (DeRISK) Center supporting N.M.H. This article has not been subjected to any EPA review and therefore does not necessarily reflect the views of the EPA. The EPA does not endorse any products or commercial services mentioned in this publication.

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REFERENCES

(1) Russell, K. L.; Hawksworth, A. W.; Ryan, M. A.; Strickler, J.; Irvine, M.; Hansen, C. J.; Gray, G. C.; Gaydos, J. C. Vaccinepreventable adenoviral respiratory illness in US military recruits, 1999−2004. Vaccine 2006, 24 (15), 2835−2842. (2) Kukkula, M.; Arstila, P.; Klossner, M. L.; Maunula, L.; Bonsdorff, C. H.; Jaatinen, P. Waterborne outbreak of viral gastroenteritis. Scand. J. Infect. Dis. 1997, 29 (4), 415−418. (3) Lee, S. H.; Kim, S. J. Detection of infectious enteroviruses and adenoviruses in tap water in urban areas in Korea. Water Res. 2002, 36 (1), 248−256. (4) Crabtree, K. D.; Gerba, C. P.; Rose, J. B.; Haas, C. N. Waterborne adenovirus: a risk assessment. Water Sci. Technol. 1997, 35 (11-12), 1− 6.

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DOI: 10.1021/acs.est.7b04602 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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Environmental Science & Technology (5) Gerba, C. P.; Gramos, D. M.; Nwachuku, N. Comparative inactivation of enteroviruses and adenovirus 2 by UV light. Appl. Environ. Microbiol. 2002, 68 (10), 5167−5169. (6) Thompson, S. S.; Jackson, J. L.; Suva-Castillo, M.; Yanko, W. A.; El Jack, Z.; Kuo, J.; Chen, C. L.; Williams, F. P.; Schnurr, D. P. Detection of infectious human adenoviruses in tertiary-treated and ultraviolet-disinfected wastewater. Water Environ. Res. 2003, 75 (2), 163−170. (7) Shin, G.; Linden, K. G.; Sobsey, M. D. Low pressure ultraviolet inactivation of pathogenic enteric viruses and bacteriophages. J. Environ. Eng. Sci. 2005, 4, S7−S11. (8) Linden, K. G.; Shin, G. A.; Lee, J. K.; Scheible, K.; Shen, C. Y.; Posy, P. Demonstrating 4-log adenovirus inactivation in a mediumpressure UV disinfection reactor. J. Am. Water Works Assoc. 2009, 101 (4), 90−99. (9) UV Disinfection Guidance Manual; EPA 815-D-03-007; Office of Water, U.S. Environmental Protection Agency: Washington, DC, 2003. (10) Ultraviolet Disinfection Guidance Manual for the Final Long Term 2 Enhanced Surface Water Treatment Rule; U.S. Environmental Protection Agency: Washington, DC, 2006. (11) Linden, K. G.; Thurston, J.; Schaefer, R.; Malley, J. P., Jr. Enhanced UV Inactivation of Adenovirus under Polychromatic UV Lamps. Appl. Environ. Microbiol. 2007, 73 (23), 7571−7574. (12) 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. (13) Guo, H.; Chu, X.; Hu, J. Effect of Host Cells on Low- and Medium-Pressure UV Inactivation of Adenoviruses. Appl. Environ. Microbiol. 2010, 76 (21), 7068−7075. (14) Rodríguez, R. A.; Bounty, S.; Linden, K. G. Long-range Quantitative PCR for Determining Inactivation of Adenovirus 2 by Ultraviolet Light. J. Appl. Microbiol. 2013, 114 (6), 1854−1865. (15) Eischeid, A. C.; Meyer, J. N.; Linden, K. G. UV Disinfection of Adenoviruses: Molecular Indications of DNA Damage Efficiency. Appl. Environ. Microbiol. 2009, 75 (1), 23−28. (16) Beck, S. E.; Rodriguez, R. A.; Linden, K. G.; Hargy, T. M.; Larason, T. C.; Wright, H. B. Wavelength dependent UV inactivation and DNA damage of adenovirus as measured by cell culture infectivity and long range quantitative PCR. Environ. Sci. Technol. 2014, 48, 591− 598. (17) Eischeid, A. C.; Linden, K. G. Molecular Indications of Protein Damage in Adenoviruses after UV Disinfection. Appl. Environ. Microbiol. 2011, 77 (3), 1145−1147. (18) Russell, W. C. Adenoviruses: update on structure and function. J. Gen. Virol. 2009, 90, 1−20. (19) Wickham, T. J.; Mathias, P.; Cheresh, D. A.; Nemerow, G. R. Integrins alpha v beta 3 and alpha v beta 5 promote adenovirus internalization but not virus attachment. Cell 1993, 73 (2), 309−319. (20) Salone, B.; Martina, Y.; Piersanti, S.; Cundari, E.; Cherubini, G.; Franqueville, L.; Failla, C.; Boulanger, P.; Saggio, I. Integrin α3B1 is an alternative cellular receptor for adenovirus serotype 5. J. Virol. 2003, 77 (24), 13448−13454. (21) San Martín, C. Latest Insights on Adenovirus Structure and Assembly. Viruses 2012, 4 (12), 847−877. (22) Adenoviruses: Basic Biology to Gene Therapy; Seth, P., Ed.; R. G. Landes Company: Austin, TX, 1999; pp 31−38. (23) Lui, G. Y.; Roser, D.; Corkish, R.; Ashbolt, N.; Stuetz, R. Pointof-use water disinfection using ultraviolet and visible light-emitting diodes. Sci. Total Environ. 2016, 553, 626−635. (24) Oguma, K.; Rattanakul, S.; Bolton, J. Application of UV light emitting diodes to aenovirus in water. J. Environ. Eng. 2016, 142 (3), 04015082. (25) Song, K.; Mohseni, M.; Taghipour, F. Application of ultraviolet light-emitting diodes (UV-LEDs) for water disinfection: a review. Water Res. 2016, 94, 341−349. (26) Beck, S. E.; Ryu, H.; Boczek, L. A.; Cashdollar, J. L.; Jeanis, K. M.; Rosenblum, J. S.; Lawal, O. R.; Linden, K. G. Evaluating UV-C

LED disinfection performance and investigating potential dual wavelength synergy. Water Res. 2017, 109, 207−216. (27) McMinn, B. R.; Cashdollar, J. L.; Grimm, A. C.; Fout, G. S. Evaluation of the celite secondary concentration procedure and an alternate elution buffer for the recovery of enteric adenoviruses 40 and 41. J. Virol. Methods 2012, 179 (2), 423−428. (28) Cashdollar, J. L.; Brinkman, N. E.; Griffin, S. M.; McMinn, B. R.; Rhodes, E. R.; Varughese, E. A.; Grimm, A. C.; Parshionikar, S. U.; Wymer, L.; Fout, G. S. Development and Evaluation of EPA Method 1615 for Detection of Enterovirus and Norovirus in Water. Appl. Environ. Microbiol. 2013, 79, 215−223. (29) Cashdollar, J. L.; Huff, E.; Ryu, H.; Grimm, A. C. The influence of incubation time on adenovirus quantitation in A549 cells by most probable number. J. Virol. Methods 2016, 237, 200−203. (30) Bolton, J. R.; Linden, K. G. Standardization of methods for fluence (UV dose) determination in bench-scale UV experiments. J. Environ. Eng. 2003, 129 (3), 209−215. (31) Jagger, J. Introduction to Research in Ultraviolet Photobiology; Prentice-Hall: Englewood Cliffs, NJ, 1967; p 164. (32) Harm, W. Biological Effects of Ultraviolet Radiation; Cambridge University Press: New York, 1980; p 216. (33) 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. (34) Linden, K. G.; Darby, J. L. Estimating effective germicidal dose from medium pressure UV lamps. J. Environ. Eng. 1997, 123 (11), 1142−1149. (35) van Oostrom, J.; Burnett, R. M. Molecular composition of the adenovirus type 2 virion. J. Virol. 1985, 56 (2), 439−448. (36) Rux, J. R.; Burnett, R. M. Adenovirus capsid proteins. In Adenoviruses: Basic Biology to Gene Therapy; Seth, P., Ed.; R. G. Landes Company: Austin, TX, 1999; pp 5−16. (37) Vellinga, J.; Van der Heijdt, S.; Hoeben, R. C. The adenovirus capsid: major progress in minor proteins. J. Gen. Virol. 2005, 86, 1581−1588. (38) Maizel, J. V., Jr.; White, D. O.; Scharff, M. D. The polypeptides of adenovirus. 1. Evidence for multiple protein components in the virion and a comparison of types 2, 7A, and 12. Virology 1968, 36, 115−125. (39) UniProt Protein Database, 2017. http://www.uniprot.org/ uniprot/?query=j01917&sort=score (accessed June 16, 2017). (40) Nermut, M. V. Structural elements in adenovirus cores: evidence for a “core shell” and linear structures in “relaxed” cores. Arch. Virol. 1979, 62, 101−116. (41) Ostapchuk, P.; Almond, M.; Hearing, P. Characterization of Empty Adenovirus Particles Assembled in the Absence of a Functional Adenovirus IVa2 Protein. J. Virol. 2011, 85 (11), 5524−5531. (42) Wang, S. S.-S.; Wen, W.-S. Examining the influence of ultraviolet C irradiation on recombinant human γD-crystallin. Mol. Vis. 2010, 16, 2777−2790. (43) Sangsanont, J.; Katayama, H.; Kurisu, F.; Furumai, H. CapsidDamaging Effects of UV Irradiation as Measured by Quantitative PCR Coupled with Ethidium Monoazide Treatment. Food Environ. Virol. 2014, 6, 269−275. (44) Schmid, F. X. Biological macromolecules: UV−visible spectrophotometry. In Encyclopedia of Life Sciences; Macmillan: London, 2001. (45) Aitken, A.; Learmonth, M. P. Protein determination by UV absorption. In The Protein Protocols Handbook, 3rd ed.; Walker, J. M., Ed.; Humana Press: New York, 2001; pp 3−6. (46) Wigginton, K. R.; Menin, L.; Montoya, J. P.; Kohn, T. Oxidation of virus proteins during UV254 and singlet oxygen mediated inactivation. Environ. Sci. Technol. 2010, 44, 5437−5443. (47) Bosshard, F.; Armand, F.; Hamelin, R.; Kohn, T. Mechanisms of human adenovirus inactivation by sunlight and UVC light as examined by quantitative PCR and quantitative proteomics. Appl. Environ. Microbiol. 2013, 79 (4), 1325−1332.

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DOI: 10.1021/acs.est.7b04602 Environ. Sci. Technol. XXXX, XXX, XXX−XXX