Adenovirus Replication Cycle Disruption from Exposure to

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Adenovirus Replication Cycle Disruption from Exposure to Polychromatic UV Irradiation Bernardo Vazquez-Bravo, Kelley Goncalves, Joanna L. Shisler, and Benito J. Marinas Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b06082 • Publication Date (Web): 26 Feb 2018 Downloaded from http://pubs.acs.org on March 2, 2018

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Environmental Science & Technology

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Adenovirus Replication Cycle Disruption from Exposure to Polychromatic UV Irradiation

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Bernardo Vazquez-Bravo1, 3, Kelley Gonçalves2, 3, Joanna L. Shisler2, 3, Benito J. Mariñas1, 3,*

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Department of Civil and Environmental Engineering, 2Department of Microbiology and College

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of Medicine, and 3Safe Global Water Institute, University of Illinois at Urbana-Champaign,

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Urbana, Illinois 61801, United States

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ABSTRACT

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Polychromatic ultraviolet (UV) light with bandwidth of 20 nm and peak emission centered at

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224, 254, or 280 nm (UV224, UV254, and UV280) were used to inactivate human adenovirus type 2

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(HAdV-2). Quantitative Polymerase Chain Reaction (qPCR), and Reverse Transcriptase qPCR

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assays were used to elucidate the step in the HAdV-2 replication cycle that was disrupted after

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UV exposure. UV treatment at any of the wavelengths analyzed did not inhibit association of

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HAdV-2 to the host cells even after exposure to a fluence (UV dose) that would produce a virus

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inactivation efficiency, measured by plaque assay, greater than 99.99 %. In contrast, UV

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irradiation at all three peak emissions disrupted early E1A gene transcription and viral DNA

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replication, but different mechanisms appeared to be dominating such disruptions. UV224

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seemed to have little effect on the integrity of the viral genome but produced a structural

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transformation of the viral capsid that may inhibit the delivery of viral genome into the host cell

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nucleus. On the other hand, UV254 and UV280 did not affect the integrity of the viral capsid but

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the mutations they produced on the viral genome might cause the inhibition of the early gene

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transcription and DNA replication after the viral genome successfully translocated into the host

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cell nucleus.

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INTRODUCTION

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Water disinfection with ultraviolet (UV) light is gaining popularity due to its effectiveness to

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inactivate chlorine-resistant pathogens, while forming lower levels of toxic disinfection by-

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products (DBPs) compared to chlorination, the most common water disinfection approach

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currently used throughout the world1,2. Two common types of UV light sources for water

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disinfection are monochromatic low-pressure (LP) and polychromatic medium-pressure (MP)

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UV mercury-vapor lamps. LP UV lamps emit light at 254 nm, whereas MP UV lamps emit light

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with a wide wavelength range including those (200-300 nm) with germicidal properties2.

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Adenovirus is a waterborne pathogen of concern because it has the highest resistance to LP UV

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inactivation among all waterborne pathogens found in drinking water and thus it is considered a

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benchmark in regulations3,4,5. Adenovirus is a non-enveloped, icosahedral, double-stranded (ds)

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DNA virus with a capsid or protein coat surrounding the genome. There are more than 50

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adenovirus serotypes causing different diseases6 and many of them have been found in

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recreational and drinking water sources around the world3,7,8,9. Due to its known public health

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risk, adenovirus has been included in the U.S. Environmental Protection Agency (USEPA)’s

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Contaminant Candidate List Four (CCL 4)10. The USEPA established a fluence of 186 mJ/cm2

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required for a 4-log inactivation of viruses based on the resistance of adenovirus to LP UV light4.

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MP UV light is found to be more effective than LP UV light at inactivating adenovirus when

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using tissue culture systems to assess virus replication11-14. Polymerase Chain Reaction (PCR) or


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quantitative PCR (qPCR) assays have shown that both LP UV and MP UV irradiation have

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similar capacity to damage the adenovirus genome, and yet infectivity assays reveal the

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occurrence of different inactivation kinetics with the two light sources15,16. It has been suggested

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that irradiation emitted by a MP UV lamps inactivates adenoviruses by damaging the viral

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genome, the viral proteins, or both14,15. However, the actual mechanisms by which

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polychromatic MP UV inactivates adenovirus are unknown. Because different wavelengths

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could preferentially target different regions of the genome or proteins in the capsid, it is

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important to elucidate the mechanisms of inactivation by different wavelengths within the

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germicidal range of 200-300 nm. For these reasons, we chose to examine the effect that specific

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wavelength have on several steps of the viral replication cycle that might render the virus non-

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infectious.

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Adenovirus has a dsDNA genome with ~36 kb pairs surrounded by an icosahedral capsid with

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240 hexon protein trimers, 12 pentameric penton proteins at each vertex, and 12 fiber protein

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trimers protruding from each penton. Adenovirus uses its fiber-like projections to attach to a

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host cell receptor such as the coxsackie-adenovirus receptor (CAR)17. Next, the RGD loop

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domains of the penton base protein attach to ανβ integrins in the host cell17,18, triggering viral

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internalization by endocytosis18. Inside the endosomes the vertices of the viral particle

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dissociate, protein VI lyses the endosome, releasing the viral particle into the cytosol17. The viral

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particle interact with microtubules transporting them toward the nucleus17. Upon reaching the

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nuclear membrane the viral particle further disassembles and by ~2 h post infection (p.i.) the

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viral DNA and associated protein VII is imported into the nucleus17,18. Inside the nucleus, the

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E1A gene quickly undergoes transcription, followed by genome replication, which initiates about



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6 h after infection, approximately at the same time as late genes transcription to produce

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structural proteins such as hexon. Typically, by 48 h p.i. a productive viral replication cycle is

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completed17,19.

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Ethidium monoazide bromide (EMA) (Invitrogen, ThermoFisher Scientific, Inc., Waltham, MA)

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was used to analyze if UV irradiation affected the integrity of the adenovirus capsid. EMA is a

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fluorescent nucleic acid stain that covalently binds to nucleic acids after exposure to visible

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light20. When EMA binds to the viral genome, the affected genome region does not amplify by

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qPCR. EMA can only access the adenovirus DNA if the capsid has undergone a certain level of

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disruption. Therefore, PCR not amplifying the adenovirus DNA would signal damage of the

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adenovirus capsid to the extent that EMA internalizes after UV treatment. Previous work used

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qPCR coupled with EMA to assess the capsid integrity of HAdV-5 after exposure to UV

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irradiation with peak emissions in the 240-320 nm range21. Our research expands on this work

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by assessing narrower bandwidths within that wavelength range as well as the region of high UV

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absorbance by proteins (wavelength < 240 nm).

85 86

Adenovirus inactivation experiments performed at different UV wavelengths served as a baseline

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for our novel molecular work assessing the inhibition of the adenovirus replication cycle. This

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analysis was important for better understanding the details of the inactivation capacity of MP UV

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compared to that of LP UV. qPCR, and Reverse Transcriptase qPCR (RT-qPCR) were used to

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assess disruption of genome replication, and early and late mRNA transcription. These findings

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can be the foundation to create more effective and efficient disinfection strategies that

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incorporate UV technologies. Additionally, by knowing what transformations result from


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exposure to different UV wavelengths, molecular sensors could be designed to distinguish

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transformed targets from those untransformed and therefore differentiate between infectious and

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non-infectious viruses. The major objective of this research is to elucidate using molecular

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techniques what steps in the adenovirus replication cycle were disrupted after exposure to UV

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radiation emitted in different regions of the MP UV spectrum.

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MATERIALS AND METHODS

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Ultraviolet Light Irradiation Experiments

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UV inactivation experiments were performed using a 1-kW Rayox collimated system operating

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with a polychromatic MP UV lamp (A300014, Calgon Carbon Co., Pittsburgh, PA). Bandpass

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filters (Andover Corporation, Salem, NH) with maximum peak transmittance at 224, 254, and

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280 nm, and bandwidth of about 10 nm at half of the maximum peak transmittance were used to

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isolate specific regions of UV radiation emitted by a polychromatic MP UV lamp. These non-

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overlapping spectra were selected to represent different inactivation kinetics observed for three

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distinct regions within the germicidal range of 200-300 nm after performing preliminary

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experiments with thirteen bandpass filters with maximum peak transmittance within the 218-297

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nm range (Figure S1 of SI). Each filter was installed at the end of the collimator, and the

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spectral irradiance of the light passing through the filter was measured with a BLK-C-50

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spectrometer (StellarNet Inc. Tampa, FL). The light intensity distribution across the irradiated

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water surface was measured with a 1400A radiometer and SEL 240 detector (International Light,

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Peabody, Massachusetts). The spectral irradiance was used as the weighting function to

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calculate the average fluence rate in the water sample following the standard protocol for a

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bench-scale UV apparatus using a polychromatic UV source by Bolton and Linden22. The



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radiometer readings were used to adjust for the non-uniformity of the light distribution across the

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irradiated surface (petri factor). The average fluence rate calculations accounted for the variation

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of the sensitivity of the SEL 240 detector to measure the irradiance (sensor factor), and the UV

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absorbance between 200 and 300 nm of the water sample (UV-2700 Shimadzu

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spectrophotometer) adjusting for the water sample depth (water factor), distance of the

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suspension from the UV lamp (divergence factor), and water surface reflection (reflection

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factor). A recirculating water-jacketed reactor (23ᵒC) containing 20 mL of 1 mM carbonate

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buffer solution (CBS, Fisher Scientific) in MilliQ water was dosed with HAdV-2 at

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concentrations of (6-20)×105 plaque forming units (PFU) per mL and irradiated by UV light at

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the different band ranges. The fluence (UV dose) applied to HAdV-2 samples was controlled by

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varying the exposure time. All experiments were performed in duplicate. Additional details are

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available in Text S1 of supporting information (SI).

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Virus Propagation and Infectivity Assessment

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Human lung A549 carcinoma cells (CCL-185) and human adenovirus type 2 (VR-846) were

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obtained from the American Type Culture Collection (Manassas, VA). A549 cells were used for

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adenovirus propagation and infectivity assessment with a soft agar overlay plaque assay. Cell

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culture growth, virus propagation, and infectivity assessment assays were performed following

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previously reported protocols1,23,24.

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Amplicon Integrity

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To assess genome damage after UV light treatment, viral DNA was extracted from UV-treated

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and untreated virions following the extraction protocol for the DNeasy Blood & Tissue Kit


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(Qiagen, Valencia, CA). Primers previously designed to amplify different regions of the

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adenovirus genome16,24 were used to assess if such regions were amplifiable by qPCR after UV

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light exposure. The genome regions selected represent different steps during the replication

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cycle; the E1A gene is the first gene transcribed during an early phase, and the hexon gene is a

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late gene that is transcribed soon after viral genome replication begins17. A 0.1-kilobase (kb)

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portion each of the E1A (nucleotides 673-778) and hexon (nucleotides 19,400-19,510) viral

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genes was subjected to qPCR amplification. Additionally, a 1.1-kb fragment (nucleotides

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17,822-18,996) was PCR-amplified to target a larger section of the genome to compare the UV-

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induced effect observed with the 0.1-kb E1A and hexon amplicons. Details about the procedures

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followed and primer sets used are in Text S2 and Table S1 of SI. Absolute copy numbers of the

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E1A and hexon amplicons, as well as the 1.1-kb fragment were determined using the standard

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curves described in Text S5 of SI.

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Ethidium Monoazide Exposure

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The inhibition of qPCR amplification due to EMA-DNA interactions was assessed by measuring

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the ratio of amplified DNA fragments present in UV-treated samples as compared to those

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present in the untreated sample. Protocols followed were from previous studies20,21,25 with

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modifications detailed in Text S3 of SI.

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Viral Genome Replication in Host Cells and mRNA Transcription

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HAdV-2 genome replication and mRNA transcription was quantified using previously reported

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protocols24 (see Text S4 of SI). Briefly, viruses were treated with light at each UV bandwidth,

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and then inoculated onto cell host monolayers. After incubation for 90 min at 4 ᵒC the inocula



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was removed and the monolayers were washed twice with 1 mL of ice-cold phosphate buffer

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saline (PBS) to remove unbound viruses. Viral genome replication and mRNA transcription in

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infected cells were monitored in the subsequent 48 h, a time by which one full replication cycle

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should occur17. Total DNA or RNA was extracted from the A549 cellular monolayers with

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bound HAdV-2 at various times post-infection (0, 4, 12, 24, 36, and 48 h). qPCR was used to

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quantify 0.1-kb amplicons of the E1A and hexon genes as proxy of the whole genome (primer

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sets listed in Table S1 of SI). The absolute copy numbers of viral E1A and hexon qPCR

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products was normalized per 100 β-actin copies and determined using the standard curves

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described in Text S5 of SI. Cells infected with untreated virus sample were used as control. At

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time 0 h p.i. the amplicon copies detected would correspond to attached viruses. RNA samples

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were reverse transcribed by PCR into cDNA before they were analyzed by qPCR (RT-qPCR)

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with the same primer sets used for DNA extracted samples to quantify E1A and hexon mRNA

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transcripts.

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qPCR Data Analysis and Statistical Analysis

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Quantification cycle (Cq) values obtained from the qPCR or RT-qPCR assays were used to

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determine the absolute copy numbers of each amplicon based on standard curves generated by

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amplifying 10-fold serial dilutions of DNA positive controls as previously described16,24 (see also

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Text S5 of SI). Analysis of covariance (ANCOVA) was used to compare the linear regression

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slopes and intercepts between relative decrease in DNA amplification, genome replication, or

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mRNA transcription, with the virus inactivation assessed by plaque assay in order to determine if

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they were statistically similar (P > 0.05). Absolute copy numbers of genome fragments

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amplified by qPCR were analyzed using a t test (one-tailed with unequal variances) to determine


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if genomic amplicons detected in UV-treated samples were statistically different to those in the

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untreated sample.

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RESULTS AND DISCUSSION

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Inactivation Kinetics of HAdV-2 Exposed to UV Irradiation

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The sections of the MP UV spectra covered with each bandpass filter, and the corresponding

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inactivation kinetics of HAdV-2 are shown in Figure S1 of SI. In general the observed

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inactivation kinetics are in agreement with what was previously reported14 using UV light

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emitted by a tunable laser adjusted at a similar set of wavelengths to inactivate HAdV-2. Both

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studies show similar tendencies contrasting the inactivation efficiency between UV irradiation

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emitted below and above 240 nm. This is confirmation that the enhanced effectiveness of MP

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UV light to inactivate HAdV is possibly due to protein damage by the lower wavelengths given

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that DNA absorption of UV light at wavelengths below 240 nm does not follow the action

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spectrum for adenovirus14. The differences in the viral sensitivity at some of the nominal peak

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emissions between both studies could be due to the different UV light sources utilized (highly

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monochromatic irradiation provided by the laser-UV emitter compared to narrow polychromatic

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irradiation using the bandpass filters in this study). Wright et al.26 also reported differences in

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the dose response of bacteriophages obtained by filtered MP UV light using a 254 nm bandpass

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filter compared to the response obtained using a monochromatic LP UV lamp. This reproduction

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of results was important to confirm consistency with the published data, and to serve as basis for

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the novel molecular work on the adenovirus replication cycle that is the main objective of this

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study. The analysis subsequently presented for the UV irradiation at the three regions selected



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will be referred to as the nominal peak transmittance of the filter used and indicated as UV224,

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UV254, and UV280.

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Pseudo first-order inactivation kinetics was used for analysis and the results were consistent with

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what was reported previously14, UV224 was most effective to inactivate HAdV-2 among the three

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wavelength ranges analyzed. UV224 (k=0.181 cm2/mJ, R2=0.995) required a fluence of ~20

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mJ/cm2 to reach a HAdV-2 survival ratio N/N0 = 10-4, which corresponds to a 4-log (99.99%)

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adenovirus inactivation, as detected by plaque assay. In contrast, UV254 (k=0.032 cm2/mJ,

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R2=0.994) showed slower kinetics requiring a fluence of ~125 mJ/cm2 to achieve 4-log

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adenovirus inactivation as detected by plaque assay. For UV280 (k=0.036 cm2/mJ, R2=0.986) an

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intermediate fluence of ~110 mJ/cm2 was required to achieve 4-log HAdV-2 inactivation.

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ANCOVA determined that the linear regressions of the three wavelength ranges were

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significantly different (P < 0.05). Figure S2 of SI shows the sections of the MP UV spectra

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covered using the UV224, UV254, and UV280 bandpass filters, and their corresponding inactivation

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kinetics of HAdV-2 obtained in this study.

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Effect of UV Light on Genomic DNA Amplification and Capsid Integrity

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To understand the molecular basis of virus disinfection at each wavelength range used above, we

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first examined the extent of viral genome and capsid damage induced by the three UV

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wavelengths investigated in purified virions independent of infection. Figure S3 of SI displays

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the combined ratios of E1A and hexon amplicons from UV-treated samples compared to the

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untreated samples (0.1-kb/0.1-kb0) for UV224, UV254, and UV280. For all the three wavelength

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ranges, relatively similar copy numbers of the E1A and hexon amplicons were detected in the


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UV-treated and untreated HAdV-2 samples (P > 0.05, t test). These data suggest that the 0.1-kb

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amplicons of E1A and hexon genes did not change significantly within the light fluence ranges

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investigated. However, it is important to note that these amplicons cover about 12.1% and 3.8%

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of the whole E1A and hexon genes, respectively, and damage to other regions of these genes

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could occur. Next, we further assessed genome damage using a 1.1-kb DNA fragment following

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the protocols previously reported14,16, and the results (Figure S4 of SI) indicated that genome

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damage increased with treatment according to UV224 0.05). Collectively, these results indicated that

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UV224, UV254, and UV280, interrupted DNA synthesis or an earlier step in the replication cycle,

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but did not inhibit virus association to the host cells. As expected, the relative quantification data

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for the hexon gene, shown in Figure S7 of SI, were similar to those for the E1A gene, supporting

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that the step inhibited was earlier in the replication cycle.

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Effect of UV Light on E1A mRNA Synthesis

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Since DNA replication was disrupted, the effect of UV exposure on early gene transcription

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(E1A mRNA synthesis), which occurs prior to DNA synthesis, was analyzed next. Once again,

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HAdV-2 samples were incubated with host cells after various levels of exposure to UV

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irradiation at each target wavelength. At different times post-infection, cells were harvested and

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transcription of the E1A gene was detected using RT-qPCR. The numbers of E1A mRNA

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determined for each wavelength as a function of fluence and p.i. time are shown in Figure S8 of

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SI.

290 291

The relative ratios of E1A mRNA transcripts from UV-treated samples compared to the

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untreated control (E1A/E1A0) by the end of one viral replication cycle (48 h p.i.) quantified for

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each wavelength are displayed and compared to the decrease in virus infectivity measured by

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plaque assay (N/N0) in Figure 2. ANCOVA determined that the linear regression slopes and

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intercepts between the relative expression levels of E1A mRNA and the reduction in viral

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infectivity (N/N0) assessed by plaque assay were not significantly different (P > 0.05) for UV224.

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This implied that UV224 inhibited adenovirus infection by blocking early E1A gene transcription

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or a prior step in the replication cycle.



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In contrast, ANCOVA determined that the linear regressions of the relative expression of E1A

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mRNA synthesis for UV254 and UV280 did not match the corresponding virus inactivation levels

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(N/N0) (P < 0.05 at both wavelengths). UV254 and UV280 produced a greater decrease in viral

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inactivation and relative DNA replication compared to the decrease in E1A mRNA synthesis. It

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is likely that at these two wavelength ranges UV-induced mutations in the viral genome template

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from targeted regions that are not transcribed in the early phase of the replication cycle are

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preventing DNA synthesis, and resulting in the higher decrease in DNA replication and

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infectivity observed.

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Occurrence of DNA Damage Repair

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Although a decrease in hexon mRNA synthesis was expected, since it occurs at a similar time or

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shortly after DNA replication begins17, the effect of UV treatment on this step was also assessed

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and the results are included in Figure S9 of SI (absolute values) and Figure 3 (relative ratios). A

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comparison of data sets in Figures 3 and S7 of SI reveals that by 48 h p.i. for UV224 treatment, as

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expected, the hexon mRNA transcription data match the results obtained for hexon DNA

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amplicon replication. In contrast, ANCOVA determined that the relative levels of hexon mRNA

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transcription were significantly lower compared to those for hexon amplicon replication for both

317

UV254 and UV280 treatment were significantly different (P < 0.05), with the discrepancy for

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UV280 being more pronounced. One possible explanation for these differences in hexon mRNA

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transcription and hexon DNA replication observed by 48 h p.i. could be a delay associated with

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genome repair. An interesting finding was observed when the data for hexon mRNA

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transcription at various times post-infection prior to 48 hours were plotted in relative


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quantification format as shown in Figure 3. A delayed increase in hexon mRNA synthesis is

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observed in UV treated viruses, particularly for the two wavelengths where genome damage was

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prevalent (UV254 and UV280), which might be an indirect indication of genome repair taking

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place. Relatively speaking, UV224 (which produced the least damage to the viral genome from

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the three wavelength ranges analyzed by a fluence that caused a 4-log HAdV-2 inactivation)

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approached the reduction in infectivity assessed by plaque assay more readily. For the UV254

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hexon mRNA transcription data, delays were apparent with respect to the 24 and 36 h p.i. curves

329

at UV224 even though the transcription data nearly reached the viral inactivation curve by p.i. of

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48 h. The levels of hexon mRNA transcription for UV280 were generally lower than those for

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UV254 and by 48 h p.i. the expression levels of hexon mRNA did not match the viral inactivation

332

curve. It is likely that longer post-infection time would be required for viruses exposed to UV280

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to produce the same relative levels of hexon mRNA copies as those observed for the genome

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copies and infectious viruses. The hexon mRNA synthesis data suggests that there might be

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damage and repair of other genome regions not used in this study but important for hexon

336

mRNA synthesis. These observations highlight the need to look at other portions of the genome

337

that are responsible for late mRNA transcription. One possibility is assessing the integrity of the

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genome region encoding the major late promoter (MLP), which is situated at an intermediate

339

location between the two regions analyzed in this study (E1A and hexon genes), and from which

340

most late mRNAs originate17.

341 342

Similar trends were observed when the sets of DNA replication at post-infection times prior to

343

48 h p.i. were plotted in relative quantification format as shown in Figure S10 of SI. UV254 and

344

UV280 seemed to require longer time than UV224 to reach the level of inactivation determined by



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plaque assay. This observation might be additional indication of genome repair taking place in

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the virus samples treated with UV254 and UV280. In the relative quantification plots of the DNA

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replication data (Figure S10 of SI) the samples included were those expressing genome copies to

348

a level above the background baseline E1A and hexon DNA copies that would correspond to

349

attached viruses (equivalent to time 0 h p.i. shown in Figure S5 and S6 of SI).

350 351

Finally, a comparison of the relative quantification of E1A mRNA synthesis at the different p.i.

352

times assessed for the three wavelength ranges investigated is shown in Figure S11 of SI.

353

Differently to DNA replication and hexon mRNA synthesis, the inhibition levels of E1A mRNA

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synthesis attained by increasing fluences in general did not significantly change at times

355

investigated prior to 48 h p.i., suggesting that genome repair might be happening predominantly

356

after E1A mRNA transcription takes place (likely during DNA replication). As a whole, these

357

observations suggest that different parts of the genome might have different sensitivities to

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damage induced by UV light at a particular wavelength range, and that such damage might not

359

be equally repaired in all regions of the genome that is affected. DNA damage repair during cell

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culture infectivity assays has been suggested as a possible explanation of the different adenovirus

361

inactivation effectiveness of LP UV and MP UV light13,15. Reports that quantum yields of the

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reduction in viral infectivity for double-stranded nucleic acid viruses are an order of magnitude

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lower compared to quantum yields for the formation of UV photoproducts of pyrimidines

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support the occurrence of repair in host cells28. Single and/or double-strand breaks, and DNA-

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DNA cross-links can also happen but they are about 1000 times less likely to occur compared to

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pyrimidine dimers4. UV absorption of nucleotides is predominant at intermediate wavelengths,

367

while proteins dominate the absorbance of UV light at wavelength below and above this


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intermediate range29 (see Figure S2 of SI showing intermediate range at 243-285 nm). This is

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consistent with our results at UV254 and UV280 having pronounced delays in genome replication

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and hexon mRNA synthesis. Different sensitivities of viruses to UV light has been attributed to

371

differences in viral protein sensitivity and the fact that photons absorbed at different wavelengths

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in the germicidal range does not have the same inactivation quantum yield28. Mattle et al.,30

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discussed the inactivation quantum yield by sunlight (290-320 nm) for HAdV-2 indicating that

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although the adenovirus genome readily absorbs light in the UVB range, the low inactivation

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quantum yield from UV exposure could be possible given that adenovirus could repair UV-

376

induced genome damage inside the host. Our results provide valuable insight on genome repair

377

taking place after UV exposure to different UV wavelengths and this aspect should be further

378

investigated in future studies.

379 380

Mechanistic Considerations

381

Collectively, the data obtained in this study suggests that different molecular mechanisms are

382

involved in the HAdV-2 inactivation levels achieved by the different UV wavelength ranges

383

analyzed. UV224 disinfection did not inhibit HAdV-2-host cell binding and had the lowest effect

384

on the integrity of the viral genome among the three wavelength ranges analyzed, but hampered

385

viral gene transcription and viral genome replication. In addition, assays using EMA suggested

386

that UV224 modified the viral capsid (see Figure S3 of SI). UV224 treatment of adenovirus

387

resulted in conformational changes in the virus capsid that do not preclude binding to the host

388

cell but prevent one of the subsequent steps of the infection cycle such as internalization to the

389

cytoplasm, endosomal release, or genome translocation into the host cell nucleus. The

390

adenovirus capsid is primarily formed by three major capsid proteins (hexon, fiber, and penton



Page 18 of 29

18 391

base), and several internal proteins (e.g., proteins VI and VII). One possibility is that during

392

UV224 treatment the penton base gets disrupted thus blocking internalization of the attached

393

virus. After the high-affinity binding of the fiber knob with CAR, RGD loop domains of penton

394

base subunits associate with αv integrins on the host cell with 50-fold lower affinity than the

395

interaction of fiber with CAR to promote endocytosis17. Exposure to UV224 may further inhibit a

396

successful interaction at this step if penton base is affected. Beck et al.27 indicated that a fluence

397

of 38 mJ/cm2 emitted at 220 nm reduced adenoviral penton and hexon protein quantities to ~31%

398

and ~33%, respectively of the original amounts. The authors also detected indication of protein

399

aggregation with increasing fluence at this wavelength. Their results support the hypothesis that

400

UV224 treatment might be predominantly disrupting the ability of the bound viruses to promote

401

endocytosis and internalize into the host, or that if successfully internalized it might not be able

402

to release from the endosome due to disruption of the capsid. Finally, if the virus reaches the

403

nucleus, import of the genome might be inhibited due to disruption of protein VII, since protein

404

VII mediates DNA import through the nuclear pore complex17,31. Inhibition of the virus

405

replication cycle steps post binding and prior to early gene transcription should be assessed in

406

future research to determine what viral protein modifications induced by UV224 rendered

407

adenovirus non-infectious.

408 409

UV254 treatment of viruses also allowed virus-host cell binding, but it disrupted the viral genome

410

to a greater extent than UV224 (Figure S4 of SI). The results from the EMA assay indicated that

411

UV254 did not cause modifications to the viral capsid that would allow EMA permeation (Figure

412

S3 of SI). This is consistent with the observations reported by Bosshard et al.32 in which UV254

413

did not cause degradation to structural proteins (hexon, fiber, and penton base) of HAdV-2.


Page 19 of 29


19 414

UV254 also hindered early E1A mRNA synthesis but not to the same level as the decrease in viral

415

infectivity, as was the case with UV224. For UV254 the inhibition of early gene transcription may

416

be dominated by UV-induced mutations to the template E1A gene. It is possible that the UV254-

417

treated virus could translocate its mutated genome into the cell nucleus, and E1A mRNA

418

transcription was inhibited proportionally to the level of UV-induced damage to the complete

419

E1A gene region. Likely, the viral DNA was replicated depending on the level of mutations

420

caused to the whole genome. Consequently, genome replication appeared to be the limiting step

421

that ultimately produced the reduction in infectivity assessed by plaque assay after irradiation

422

with UV254. Adenovirus may be able to repair UV-induced damage to its genome using host cell

423

repair mechanisms, such as the nucleotide excision-repair process14,15,33. Specifics about the

424

effect of viral genome repair mechanisms and their repercussions in the viral replication cycle

425

inhibitions require future exploration.

426 427

Finally, UV280 did not seem to inhibit viral binding association with the host cell either and

428

showed higher capacity than UV254 to disrupt mRNA transcription under similar applied

429

fluences. UV280 disrupted early mRNA synthesis to a greater extent than UV254, as indicated by

430

the steeper linear regression slope of the E1A mRNA synthesis plot for UV280 (slope UV280= -

431

0.027, slope UV254= -0.016) in Figure 2. Similar to UV254, UV280 did not seem to be greatly

432

affecting structural proteins as indicated by the EMA results (Figure S3 of SI). However, UV280

433

was the most effective among the three wavelength ranges analyzed in blocking qPCR

434

amplification of the 1.1-kb amplicon used (Figure S4 of SI). This suggests that the more

435

effective HAdV-2 inactivation by UV280 (compared to UV254) is likely dominated by a more

436

effective induction of mutations on the viral genome (perhaps to DNA regions associated with



Page 20 of 29

20 437

core proteins), rather than modifications to structural proteins. Three amino acids (tryptophan,

438

tyrosine, and phenylalanine) that regularly constitute proteins have UV absorption peaks at 280

439

nm; and although this peak is smaller relative to that for UV absorption of DNA, its presence

440

may be contributing to the enhanced effectiveness that UV280 showed compared to UV25429.

441

Further research is needed to elucidate if damage to a specific core protein or DNA-protein

442

complex region contributes in the viral inactivation effect observed by UV280.

443 444

Practical Implications

445

Some of the techniques utilized in this work allowed a relatively fast determination of virus

446

inactivation levels attained by UV irradiation. For all three wavelengths, results for genome

447

replication experiments by 48 h p.i. matched the decrease in virus infectivity achieved by each

448

applied fluence when infectivity was assessed by plaque assay (which required 10 days to be

449

completed). This convenient aspect could help inspect the effectiveness of water disinfection

450

approaches more rapidly when using adenovirus as the target pathogen for monitoring virus

451

inactivation by UV light.

452

The results from this study also contribute to a better understanding of the mechanisms by which

453

UV irradiation emitted at different wavelengths renders HAdV-2 non-infectious. A complete

454

understanding of this phenomenon will allow the development of more effective and efficient

455

water disinfection strategies that incorporate UV technologies, as well as rapid methods for

456

detection and monitoring of pathogens in drinking water. In addition, our results confirmed

457

better capacity of the 260-280 nm wavelength range, being UV270 the most effective (Figure S1

458

of SI), to inactivate HAdV-2 compared to UV254; making it an attractive alternative to be further

459

explored to control viruses in drinking water. This work provided insightful information toward


Page 21 of 29


21 460

a better understanding of the mechanisms by which different regions of the MP UV spectrum

461

inactivate human adenovirus during water disinfection.

462 463

ASSOCIATED CONTENT

464

Supporting information

465

Inactivation kinetics at different wavelength ranges and the sections of the MP UV spectra

466

covered with each bandpass filter; detailed protocols for the components of the materials and

467

methods section; primers used for qPCR and RT-qPCR data analysis; amplicon and capsid

468

integrity data; and the absolute and relative quantification plots for genome replication and

469

mRNA synthesis at different post-infection times for the E1A and hexon genes are included.

470

This information is available free of charge via the Internet at http://pubs.acs.org.

471 472

AUTHOR INFORMATION

473

Corresponding Author

474

*(B.J.M.) Phone: +1-217-333-6961, Email: [email protected]

475

Notes

476

The authors declare no competing financial interest.

477 478

ACKNOWLEDGMENTS

479

This research work was funded by the University of Illinois at Urbana-Champaign Institute for

480

Sustainability Energy and Environment (iSEE), the Center of Advanced Materials for the

481

Purification of Water with Systems (WaterCAMPWS), a National Science Foundation (NSF)

482

Science and Technology Center, under award no. CTS-0120978, and supported by the fellowship



Page 22 of 29

22 483

assistance from the National Council on Science and Technology (CONACYT), Mexico. Any

484

opinions, findings, and conclusions or recommendations expressed in this manuscript are those

485

of the authors and do not necessarily reflect the views of the NSF or CONACYT.


Page 23 of 29


23 REFERENCES [1] Sirikanchana, K.; Shisler, J. L.; Marinas, B. J., Effect of exposure to UV-C irradiation and monochloramine on adenovirus serotype 2 early protein expression and DNA replication. Appl. Environ. Microbiol. 2008, 74, (12), 3774-3782. [2] Eischeid, A. C.; Thurston, J. A.; Linden, K. G. UV disinfection of adenovirus: Present state of the research and future directions. Crit. Rev. Environmental Science and Technology. 2011, 41 (15), p. 1375-1396. [3] Oguma, K.; Rattanakul, S.; Bolton, J. R. Application of UV light-emitting diodes to adenovirus in water. Journal of Environmental Engineering, ASCE. 2015, ISNN 0733-9372. [4] USEPA (U.S. Environmental Protection Agency). Ultraviolet Disinfection Guidance Manual for the Final Long Term 2 Enhanced Surface Water Treatment Rule. Washington, DC. 2006. [5] USEPA (U.S. Environmental Protection Agency). National primary drinking water regulations: Ground water rule, final rule. Washington, DC. 2006. [6] Wold, W. S. M.; Ison, M. G. Adenoviruses. In Fields Virology, 6th ed.; Knipe, D. M.; Howley, P. M., Eds. Lippincott Williams & Wilkins: Philadelphia, PA, 2013; pp 1732-1767. [7] Nascimento, M. A., Magri, M. E., Schissi, C. D., & Barardi, C. R. Recombinant adenovirus as a model to evaluate the efficiency of free chlorine disinfection in filtered water samples. Virology Journal. 2015, 12, 30. http://doi.org/10.1186/s12985-015-0259-7 [8] Jiang, S. C., Human adenoviruses in water: occurrence and health implications: a critical review. Environmental science & technology 2006, 40, (23), 7132-40. [9] Rigotto, C.; Hanley, K.; Rochelle, P. A.; De Leon, R.; Barardi, C. R.; Yates, M. V., Survival of adenovirus types 2 and 41 in surface and ground waters measured by a plaque assay. Environmental science & technology 2011, 45, (9), 4145-4150. [10] USEPA. Drinking Water Contaminant Candidate List 4—Final, Federal Register /Vol. 81, No. 222 /Thursday, November 17, 2016 /Notices pp. 81099- 81114. [11] Linden, K. G.; Thurston, J.; Schaefer, R.; Malley, J. P. Enhanced UV inactivation of adenoviruses under polychromatic UV lamps. Applied and Environmental Microbiology. 2007, 73 (23), p. 7571–7574. [12] Linden, K. G.; Shin, G. A.; Lee, J. K.; Scheible, K.; Shen, C. Y.; Posy, P. Demonstrating 4log adenovirus inactivation in a medium-pressure UV disinfection reactor. American Water Works Association. Journal. 2009, 101 (4), p. 90-99.

23 ACS Paragon Plus Environment


Page 24 of 29

24 [13] 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 Science & Technology WST | 2009, 60 (4), 851-857. [14] 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. Environmental science & technology. 2014, 48, (1), 591-598. [15] Eischeid, A. C.; Meyer, J. N.; Linden, K. G. UV disinfection of adenoviruses: Molecular indications of DNA damage efficiency. Applied and Environmental Microbiology. 2009, 75 (1), p. 23–28. [16] Rodriguez, 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), 1854-65. [17] Berk, A. J. Adenoviridae. In Fields Virology, 6th ed.; Knipe, D. M.; Howley, P. M., Eds. Lippincott Williams & Wilkins: Philadelphia, PA, 2013; pp 1704-1735. [18] Greber, U. F., Nakano, M. Y., and Maarit Suomalainen, M., Adenovirus Entry into Cells, Methods in Molecular Medicine, Vol 21: Adenovirus Methods and Protocols, Edited by: W. S. M. World, Humana Press Inc., Totowa, NJ, 1999, pp 217-230. [19] Jogler C, Hoffmann D, Theegarten D, Grunwald T, Überla K, Wildner O. Replication Properties of Human Adenovirus In Vivo and in Cultures of Primary Cells from Different Animal Species. Journal of Virology. 2006; 80(7):3549-3558. doi:10.1128/JVI.80.7.35493558.2006. [20] Kim, K.; Katayama, H.; Kitajima, M.; Tohya Y.; Ohgaki, S. Development of a real-time RT-PCR assay combined with ethidium monoazide treatment for RNA viruses and its application to detect viral RNA after heat exposure. Water Science & Technology. 2011, 63 (3), p. 502-507. [21] Sangsanont, J.; Katayama, H.; Kurisu, F.; Furumai, H. Capsid-damaging effects of UV irradiation as measured by quantitative PCR coupled with ethidium monoazide treatment. Food Environ. Virol. 6 (2014), pp. 269-275. [22] Bolton, J. R.; Linden, K. G. Standardization of methods for fluence (UV dose) determination in bench-scale UV experiments. Journal of Environmental Engineering, ASCE. 2003, 129 (3), p. 209-215. [23] Page, M. A.; Shisler, J. L.; Marinas, B. J., Mechanistic aspects of adenovirus serotype 2 inactivation with free chlorine. Applied and Environmental Microbiology. 2010, 76, (9), 29462954.


Page 25 of 29


25 [24] Gall, A. M.; Shisler, J. L.; Marinas, B. J., Analysis of the viral replication cycle of adenovirus serotype 2 after inactivation by free chlorine. Environmental Science & Technology. 2015, 49, (7), 4584-4590. [25] Parshionikar, S.; Laseke, I.; Fout, G. S. Use of propidium monoazide in reverse transcriptase PCR to distinguish between infectious and noninfectious enteric viruses in water samples. Applied and Environmental Microbiology. 2010, p. 4318–4326. [26] Wright, H.B.; Mackey, E.D.; Gaithuma, D.; Fonseca, C.; Baumberger, L.; Drzurny, T.; Clancy, J.L.; Hargy, T.M.; Fallon, K.; Cabaj, A.; Schmalweisser, A.; Bierman, A.; Gribbin, C. Optimization of UV Disinfection. Awwa Research Foundation and NYSERDA, 2007. [27] Beck, S.E.; Hull, N. M.; Poepping, C.; Linden, K. G. Wavelength-Dependent Damage to Adenoviral Proteins Across the Germicidal UV Spectrum. Environ. Sci. Technol. 2018, 52, 223−229. [28] Rauth, A. M. Physical state of viral nucleic acid and sensitivity of viruses to ultraviolet light Biophysical Journal. 1965, 5 (3), 257- 273. [29] Bolton, J. R.; Cotton, C. A. The Ultraviolet Disinfection Handbook. American Water Works Association. 2008, p. 11-40. [30] Mattle, M. J.; Vione, D.; Kohn, T. Conceptual Model and Experimental Framework to Determine the Contributions of Direct and Indirect Photoreactions to the Solar Disinfection of MS2, phiX174, and Adenovirus Environ. Sci. Technol., 2015, 49 (1), pp 334–342. [31] Kremer, E. J.; Nemerow, G. R. Adenovirus tales: From the cell surface to the nuclear pore complex. PLoS Pathogens. 2015, 11(6), e1004821. [32] 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. Applied and Environmental Microbiology 2013, 79, (4), 1325-1332. [33] Guo, H. L.; Chu, X. N.; Hu, J. Y. Effect of host cells on low- and medium-pressure UV inactivation of adenoviruses. Applied and Environmental Microbiology. 2010, 76(21). p. 70687075.



Page 26 of 29

Log Reduction (N/N0, E1A/E1A0 DNA)

26

0 N/N0 DNA DNA 0 h 48 h

-1

UV224 UV254 UV280

-2 -3 -4 -5 -6 0

50

100

150

200

2

Fluence (mJ/cm ) Figure 1. The relative expression level of E1A DNA (E1A/E1A0) measured at 0 and 48 h p.i. (hours post-infection) normalized to the untreated sample is compared to the adenovirus survival ratio (N/N0) determined by plaque assay, where N is the number of infectious viruses at any given time and N0 the initial number of infectious viruses. Error bars represent the standard deviation of the mean. For each of the three conditions (UV224, UV254 and UV280) the linear regression slopes and intercepts of the relative genome replication (E1A/E1A0 DNA) at 48 h p.i. and the virus inactivation (N/N0) were not significantly different (ANCOVA, P > 0.05).


Page 27 of 29


Log Reduction (N/N0, E1A/E1A0 mRNA)

27

N/N0 mRNA 48 h

0

UV224 UV254 UV280

-1 -2 -3 -4 -5 -6 0

50

100

150

200

Fluence (mJ/cm2) Figure 2. Relative expression level of E1A mRNA synthesis (E1A/E1A0) at 48 h p.i. (hours postinfection) normalized to the untreated sample is compared to the adenovirus survival ratio (N/N0) determined by plaque assay after exposure to UV224, UV254 and UV280. Error bars represent the standard deviation of the mean. For UV254 and UV280 the linear regression of the relative mRNA synthesis (E1A/E1A0 mRNA) and the virus inactivation (N/N0) were significantly different (ANCOVA, P < 0.05).



Page 28 of 29

0

UV224

-1

UV254

N/N0 48 h 36 h 24 h

-2

0

UV280

N/N0 48 h 36 h 24 h

N/N0 48 h 36 h 24 h

-1 -2

-3

-3

-4

-4

-5

-5

-6

-6 0

10

20

30 2

Fluence (mJ/cm )

0

50

100

150 2

Fluence (mJ/cm )

0

50

100

Log Reduction N/N0, Hexon/Hexon0 mRNA

Log Reduction N/N0, Hexon/Hexon0 mRNA

28

150 2

Fluence (mJ/cm )

Figure 3. The relative expression level of Hexon mRNA synthesis (Hexon/Hexon0) at 24, 36, and 48 h p.i. (hours post-infection) normalized to the untreated sample is compared to the adenovirus survival ratio (N/N0) determined by plaque assay after exposure to UV224, UV254 and UV280. Error bars represent standard deviation of the mean.


Page 29 of 29


29 TOC Abstract Art:


Adenovirus Replication Cycle Disruption from Exposure to

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