Analysis of Hydroxyl Radicals and Inactivation Mechanisms of

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Analysis of Hydroxyl Radicals and Inactivation Mechanisms of Bacteriophage MS2 in Response to a Simultaneous Application of UV and Chlorine Surapong Rattanakul, and Kumiko Oguma Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b03394 • Publication Date (Web): 05 Dec 2016 Downloaded from http://pubs.acs.org on December 7, 2016

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Environmental Science & Technology

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Analysis of Hydroxyl Radicals and Inactivation Mechanisms of Bacteriophage MS2 in

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Response to a Simultaneous Application of UV and Chlorine

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Surapong Rattanakul and Kumiko Oguma*

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Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo,

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Japan

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*Corresponding Author: Phone/Fax: +81-3-5841-0547; e-mail: [email protected]

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Abstract

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The simultaneous application of UV and chlorine (expressed as UV/Cl2) as a water treatment

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method may be a good disinfection option for UV-resistant microorganisms, such as human

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adenoviruses (HAdVs). In this study, we developed two approaches using UV/Cl2: one to

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quantitate the OH• radicals based on the degradation of the probe compound para-

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chlorobenzoic acid (pCBA) and the other to use bacteriophage MS2 to understand the virus

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inactivation mechanisms in response to UV, chlorine and UV/Cl2 disinfection using reverse-

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transcription quantitative polymerase chain reaction (RT-qPCR), attachment and genome

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penetration assays. The results revealed that OH• radicals were produced at a concentration of

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2.70 × 10‒14 M in the UV/Cl2 treatment with a practical chlorine dose of 1 mg/L and with a

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minimum UV254 fluence of approximately 10 mJ/cm2, whereas UV or chlorine alone did not

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produce OH• radicals. In the UV/Cl2 treatment, synergistic effects on viral genome damage

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were observed, but were not directly due to OH• radicals. The ability of MS2 to penetrate the

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genome of the host bacteria was impaired, but its ability to attach to the host was not affected

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by the treatment. In conclusion, we concluded that the major cause of virus inactivation in

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response to UV/Cl2 was the damage to the viral genome caused by combination actions of

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chlorine species and OH• radicals.

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TOC/Abstract art

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INTRODUCTION

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UV technology has been shown to be an effective water disinfection method for controlling

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water-borne pathogenic bacteria and protozoa, and a fluence of 10 mJ/cm2 can achieve an

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over 4 log10 inactivation of Escherichia coli, Legionella pneumophila, Cryptosporidium

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parvum and Giardia lamblia without photoreactivation1–4. However, viruses are more

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resistant to UV than bacteria and protozoa, particularly human adenoviruses (HAdVs)5–6.

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Given the concerns regarding the public health implications of virus-borne illnesses among

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drinking water consumers, the minimum fluence required to achieve an inactivation of >4

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log10 for viruses is 186 mJ/cm2 using low-pressure UV (LPUV) under The Long-Term 2

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Enhanced Surface Water Treatment Rule7.

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Despite this, the required UV fluence for adenoviruses inactivation is very high, and a

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combination of two or more disinfectants such as UV/chlorine or UV/O3 considered as a

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multi-barrier approach for water disinfection may solve the issue. Recently the combined

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application of UV and chlorine has been increasing in water treatment plants. According to

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the results of a survey of UV implications in the United States, the orders of the combination

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treatments in water treatment plants could be UV followed by chlorine or chlorine followed

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by UV8, with the latter application also considered an advanced oxidation process (AOP)

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because an exposure of chlorine species to UV can produce radicals (OH• or Cl•) that may

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enhance the disinfection efficiency9. However, high fluences (> 100 mJ/cm2) and chlorine

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doses (> 8 mg/L) are typically employed in UV/Cl2 to achieve an AOP to remove chemical

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pollutants10–11, which raises the question of whether or not such radicals can exist at the low

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fluences and chlorine doses typically used for water disinfection. A previous study in our

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group investigated the efficacy of a simultaneous application of UV and chlorine (expressed

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as UV/Cl2) and observed synergistic viral inactivation at a practical UV fluence and chlorine


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dose of 15 mJ/cm2 and 1 mg/L, respectively implying of the enhancement inactivation by

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radicals12.

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Along with the viral control methods, virus inactivation mechanisms are also an important

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issue to understand. The infection mechanisms of viruses are complicated and are normally

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comprised of three main steps: attachment to the host, entry or genome penetration into the

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host, and genome replication in the host. During the host attachment and entry or genome

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penetration processes, viral proteins on capsid are a key component for a successive

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infection, whereas the viral genome plays an important role during replication13–14.

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Accordingly, damage to either of these viral components can inactivate the viruses by

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interrupting these steps in the infectivity processes. Although the individual effects of

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chlorine and UV on virions has been reported in many studies15–17, to date there has been no

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studies of virus inactivation mechanisms following the simultaneous application of UV and

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chlorine. Oxidants such as chlorine or ozone can potentially damage both the protein

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components of the viral capsid15 and the viral genome16, while UV light induces thymine-

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dimers in viral genomes, resulting in replication failure and could also alter protein

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components of the viral capsid17. Accordingly, it is expected that both capsid and viral

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genome can be damaged by the UV/Cl2 due to the oxidative radicals. Considering this

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background information, it is important to prove the existence of radicals during this

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treatment and also explore the fundamental virus inactivation mechanisms mediated by

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UV/Cl2 treatment to better understand the fundamental principles of the combined process.

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The objectives of this study were to (1) determine the concentration of radicals produced

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following the simultaneous application of UV/Cl2 and (2) quantify the level of each vital

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function that was lost after the UV/Cl2 treatment. An OH• radical probe compound was

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employed to investigate the occurrence of OH• radicals in the UV/Cl2 treatment under the

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same conditions used for disinfection. In addition, in order to investigate the virus 5 ACS Paragon Plus Environment


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inactivation mechanisms the, bacteriophage MS2 was used as a surrogate model of

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pathogenic viruses because of its similar morphology and infection cycles using previously

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developed quantitative PCR-based methods. We quantified the amount of OH• radicals that

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were produced and elucidated how they affected virus particles and vital functions after

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disinfection with UV/Cl2 treatment compared with each treatment individually. The results

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from this study can strengthen confidence in the combined UV/Cl2 in water treatment plants

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to the ensure safety of drinking water.

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MATERIALS AND METHODS

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Bacteriophage propagation and enumeration. Bacteriophage MS2 (ATCC 15597 B1)

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was introduced to a host cell, E. coli K-12 A/λ (F+) (ATCC 10798) obtained from the

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American Type Culture Collection (Manassas, VA) for one night and was further purified by

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chloroform extraction to obtain a stock solution for subsequent experiments. A double layer

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agar technique18 was used to assess infectivity.

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Determination of the hydroxyl radical concentrations. The concentration of OH•

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radicals was indirectly measured by observing the degradation rate of the probe compound,

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para-chlorobenzoic acid (pCBA), which has a high reactivity with OH• radicals. Briefly, a

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pCBA working solution with an initial concentration of 1.8 µM was prepared by mixing a

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100 µM pCBA stock solution with distilled water (DI, pH 7.2). The pCBA working solution

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was then exposed to 1 mg/L of free chlorine and LPUV. Samples were taken at the designated

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exposure times and immediately dechlorinated with ammonium chloride for the OH• radical

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analysis. High-performance liquid chromatography (HPLC, Dionex Ultimate® 3000, Thermo

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Scientific) equipped with a UV detector and a 3.9 × 150 mm Nova-Park® C-18 column with

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a 4-µm particle size was used to analyze the concentration of pCBA using a mobile phase

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consisted of 40 % orthophosphoric acid (pH 2.3), 40 % acetonitrile and 20 % methanol at a 6 ACS Paragon Plus Environment

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flow rate of 1 mL/min. Due to the short lifetime of OH• radicals, the concentration of OH•

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radicals at a steady-state ([OH•]ss) was calculated using Equation 1, as described in a

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previous study19:

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ୢሾ௣େ୆୅ሿ ୢ௧

=– ݇୓ୌ•,௣େ୆୅ ሾ‫݌‬CBAሿሾOH •ሿୱୱ

(Eq.1)

where kOH•,pCBA is the rate constant of the reaction of pCBA with the hydroxyl radicals (5 × 109 M–1 s–1) and [OH•]ss is the steady-state concentration of OH• radicals.

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Disinfection experiments. The virus inactivation mechanisms were investigated after three

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types of treatments: UV irradiation, chlorination and UV/Cl2, and disinfection procedures are

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explained in Text S1 in the supplement information.

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Molecular assays. Three types of molecular assay, including RT-qPCR, attachment, and

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genome penetration assays were established to assess virus inactivation mechanisms of the

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UV/Cl2 treatment. Figure 1 illustrates investigated the infection steps, goals and expected

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outcomes for each assay.

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RT-qPCR assays (RT-PCR and Real-time qPCR). The number of MS2 genome segments

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lost after each treatment method was quantified by real-time reverse transcription quantitative

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polymerase chain reaction (RT-qPCR). Briefly, total RNA was first extracted by using a

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QIAamp Viral RNA Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s

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instructions, and subsequently reverse transcribed into cDNA using a High Capacity cDNA

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Reverse Transcription Kit (Applied Biosystems, California). The cDNA solutions were

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further analyzed by qPCR assay to quantify the viral concentration, which was expressed as

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the number of genome copies. The numbers of genome copies from the untreated (N0) and

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treated samples (Nt) were used to calculate the first-order rate constant for the observed

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genome damage (kobserved_damage), including lesions caused by degradation, breakage, and the



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formation of dimers at the amplicon region after each treatment method, using the slope of

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log10(Nt/N0) versus fluence, CT, or time.

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Virus attachment assay. E. coli K12 F+ (A/λ) was incubated in Lennox L broth base (LB

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broth) (Invitrogen, USA) at 37 °C for 2.5 hrs and then centrifuged at 6,000 rpm for 10 min.

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The supernatant was removed and the pellet was re-suspended with 0.07 mM PBS, in which

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phage attachment without phage development is allowed18,

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optical density (OD600) of 0.2 cm‒1 (~1.6 × 106 CFU/mL). The E. coli suspension was mixed

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with equal volumes of the control (untreated MS2) or disinfected samples (treated MS2), with

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a multiplicity of infection (MOI, a ratio between number of viruses and host cells and details

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can be found in SI, Text S3) of 0.01 for both control and disinfected samples18. At high

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bacterial concentrations (low MOI) during the attachment process, host-attached MS2 can

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reversibly desorb18 and there is an increase in non-specific attachment, therefore a proper

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MOI value is crucial for a success of attachment assays. This was verified by results of

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preliminary experiments performed with control samples which showed that the recovery rate

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(a percentage of MS2 obtained after performing attachment assays) of specific attachment

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was highest and non-specific attachment was lowest at an MOI of 0.01 (Table S2 and S3).

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The mixed samples were incubated at 37 °C for 2 hrs to initiate the irreversible host

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attachment process onto sex-pili of E. coli, after which samples were centrifuged at 12,000

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rpm for 10 min to recover the E. coli cells. The supernatant containing freely suspended MS2

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was withdrawn and the pellet containing host-attached MS2 was re-suspended with cold PBS

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(4 °C). The phage particles were extracted from the E. coli cells using three freeze-thaw

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cycles22–23, and the lysates were then filtered through a 0.2-µm pore membrane (Advantec,

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Japan) to remove bacterial debris, a result of freeze-thaw method. The filtrate containing MS2

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from the attachment assay was further quantified with RT-qPCR assays. The first-order rate

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constant for genome loss in the attachment assay (kattachment) was calculated from the slopes of

20–21


(Figure S6), to obtain an

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the log10(Nt/N0) versus fluence, CT, or time and the first-order rate constant for the loss of

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attachment ability (k′attachment) was obtained by subtracting kattachment from kobserved_damage.

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Virus genome penetration assay. The genome penetration assay was used to measure the

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loss of the genome penetration ability of virus after attaching to the sex-pili. The control and

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disinfected samples were mixed separately with E. coli at an MOI of 0.01 and incubated on

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ice maintained at 4 °C for 5 hrs to allow MS2 to attach without the inserting their genomes

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into the host cells. Subsequently the samples were incubated at 37 °C for 2 hrs to allow the

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viruses to transfer their genomes into the host cells via the sex-pili. The samples were then

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exposed to 0.005% sodium dodecyl sulfate (SDS), a protein denaturant that can detach pili

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from bacterial cells24–26, to remove the pili-attached virus from the host cells. Samples were

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centrifuged, washed once with cold PBS18 and re-suspended in cold PBS. After treating the

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samples with SDS and washing with cold PBS, only the viral genomes that were introduced

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into the host cells should remain. The viral genomes were extracted from the host cells and

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quantified with RT-qPCR assays, as described above. The first-order rate constant for

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genome loss in the genome penetration assay (kpenetration) was calculated from the slopes of the

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log10(Nt/N0) versus fluence, CT or time and the first-order rate constant for the loss of the

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ability of the virus genome to penetrate the host (k′penetration) was obtained by subtracting

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kpenetration from kobserved_damage and k′attachment.

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Disinfection kinetics. For UV and chlorine, genome degradation/inactivation rate constants

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were determined from a first-order Chick-Watson model (Equation 2):

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logଵ଴ (ே೟ ) =– ݇‫ݐܥ‬

ே

(Eq. 2)

బ

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where, N0 is the numbers of genome copies or viruses of the untreated samples, Nt is the

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numbers of genome copies or viruses of treated samples, k is the inactivation rate or genome

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reduction rate constant, C is the free chlorine concentration or UV irradiance and t is the

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contact or exposure time.



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In UV/Cl2, the initial concentration of free chlorine and UV irradiance were the same as

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testes with either UV or chlorine alone, then genome reduction/inactivation rate constants

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were determined from the first-order Chick-Watson model regardless of disinfectant

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concentration (Equation 3):

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logଵ଴ ( ೟ ) =– ݇‫ݐ‬

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where, t is the contact time in the UV/Cl2.

ே

(Eq. 3)

ேబ

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Data analysis. The first-order rate decay constants obtained from the different assays,

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including the infectivity (kinfectivity), RT-qPCR (kobserved_damage), attachment (kattachment) and

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genome penetration assays (kpenetration), were compared with each other under that same

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treatment conditions to estimate if decay rate constants were significantly different by

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determining the significance of the differences between slopes by analysis of covariance

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(ANCOVA) using the SPSS® statistic version 17 software. A p-value less than or equal 0.05

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indicated significant differences.

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RESULTS AND DISCUSSION

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Hydroxyl radical concentrations following UV/Cl2 treatment. Because the predominant

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photo-oxidant species produced in response to chlorine photolysis is OH• radicals at pH

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higher than 127, it is assumed that degradation of pCBA by Cl• is trivial and only the OH•

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radicals were quantified in this study. The [OH•]ss was mathematically determined as

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described above. Figure 2 shows that the [OH•]ss calculated from the observed pseudo-first

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order rate of pCBA degradation in DI was 2.70 × 10–14 M. A pCBA degradation rate by direct

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UV photolysis was not considered because it can be neglected as shown in Figure S2. The

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[OH•]ss observed for the UV/Cl2 treatment in this study was relatively low compared with

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UV/H2O2 treatment28–29, which could be a result of the use of a low chlorine dose (1 mg/L)

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and a low UV irradiance (5.30 × 10–10 Einstein cm–2 s–1) as reactants required for OH• 10 ACS Paragon Plus Environment

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production in the UV/Cl2 treatment30. The theoretical [OH•]ss was determined using a model

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that considered the effects of the initial oxidant concentration and scavengers31 to confirm

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that the OH• radicals played a major role in the UV/Cl2 treatment, and the details of the

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calculation are presented in the supporting information (Text S5).

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Using the model, the theoretical [OH•]ss was 2.53 × 10‒14 M, which was subsequently used

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to calculate the trend of pCBA degradation based on exposure to the theoretical [OH•]ss. It

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was found that the observed and estimated pCBA concentrations at different fluences were

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comparable (p > 0.05), and this agreement affirmed that OH• radicals were the predominant

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species that played a major role in MS2 inactivation following UV/Cl2 treatment.

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Hydroxyl radical exposure of MS2. The profile of MS2 log10 inactivation as a result of

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OH• radicals is shown in Figure 3. The concept of OH• radical exposure is expressed as an

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OH•-CT value, the product of [OH•]ss (M) and contact time (min). The contact times of MS2

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with OH• were determined by subtracting the log10 inactivation following UV/Cl2 treatment

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from the log10 inactivation following treatment with either UV or chlorine (Table S5)

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obtained in our previous study investigating MS2 inactivation by UV/Cl2 under the same

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conditions12. According to Figure 3, the rate constant of MS2 inactivation by OH• radicals

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calculated from the slope of the regression curve was 1.07 × 1014 M‒1 min–1. This inactivation

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rate constant in response to the OH• radicals was higher than the constants from other studies,

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which reported inactivation rate constants ranging from 1.93 × 1011 – 9.30 × 1012 M–1 min‒1

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28‒29, 31

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the advanced oxidation process used to produce the OH• radicals, as previous studies

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acquired the inactivation rate constants following treatment with UV/H2O2.

. The higher inactivation rate observed in this study might be due to the differences in

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Effects of UV, chlorine, and UV/Cl2 on the viral genome. Based on the increase in the

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log10 inactivation due to the OH• radicals produced by UV/Cl2, RT-qPCR assays were applied



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to estimate the damage to the target genomes (referred to observed genome damage in later

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discussions) for samples treated with UV, chlorine, or UV/Cl2 treatment. Following UV

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treatment, the genome reduction rate constant due to genome damage (kobserved_damage) was 3.2

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± 2.9 × 10‒3 cm2/mJ (Figure S7), which was not significantly different from a slope of zero (p

265

> 0.05), implying that the UV treatment did not induce damage in the target genome. The

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result agreed with a previous study showing that the reduction in amplifiable genome was

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small compared to the reduction of infectivity after UV treatment with same fleunces32. In

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contrast, the actions of chlorine, which is an oxidant, were expected to cause more damage to

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the viral genome; indeed, an increasing trend of genome damage was observed after exposure

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to different levels of chlorine (CT), with a genome reduction rate constant of 6.4 ± 0.4 × 10‒1

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L/mg*min (Figure S8).

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In addition to the individual effects of UV and chlorine on the viral genome, the effects of

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the combination of UV and chlorine in the UV/Cl2 treatment were also investigated, as shown

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in Figure 4. Time-based genome reduction rates were determined because the initial chlorine

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concentration and UV irradiance were the same in the chlorine, UV and UV/Cl2 treatments.

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Interestingly, the genome reduction rate observed following the UV/Cl2 treatment proceeded

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at a high rate compared with the separate treatments with either chlorine or UV, and the rate

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significantly increased (p < 0.05). The results imply that observed synergistic effects

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(synergistic factor of 2.1) were a direct result of a strong oxidizing agent; therefore, the

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effects of OH• radicals on viral genome were investigated with an OH• radicals based

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process, UV/H2O2 to confirm the hypothesis. While Figure S4 shows that the MS2

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inactivation rate was enhanced by OH• radicals, viral genome reduction was not due to OH•

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radicals (Figure S5), which was consistent with a previous study investigating adenovirus

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inactivation under UV/H2O233. Based on these results, it suggests that OH• radicals may


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enhance inactivation efficacy by causing damage on the viral capsid, and possibly

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subsequently facilitated diffusion of chlorine species into the virions.

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Effects of the UV, chlorine and UV/Cl2 treatments on attachment ability. Virus

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attachment assays, as described above, were performed on treated samples and control

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samples (untreated samples) to assess effects of each treatment on virus attachment ability,

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the results of which are shown in Figure 5. The genome reduction rates measured in the

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attachment assay (kattachment) in all treatment processes were not significantly different (p >

292

0.05) with kobserved_damage resulting in negligible values of k'attachment. This result was consistent

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with a previous study investigating the loss of attachment ability of bacteriophage PR772

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after chlorination34. In addition, we elucidated that in the attachment assays only a small

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portion of recovered MS2; approximately 13% were due to non-specific interactions as

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shown in Table S3 and it can be neglected. We can conclude that the viruses were still able to

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attach to the host cells after exposure to UV, chlorine or UV/Cl2 treatment.

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Effects of UV, chlorine, and UV/Cl2 on genome penetration ability. In addition to the

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loss of attachment ability, the loss of genome penetration ability, a subsequent step in the

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virus infective cycle, was also determined. Figure 5 shows that genome reduction rate

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constants for each treatment method measured using the virus genome penetration assay

302

(kpenetration) were significantly different from kobserved_damage (p < 0.05), which allowed us to

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further calculate the actual genome reduction rate constants due to the loss of penetration

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ability, as shown in Table S5. The results suggest that the virus genome penetration ability

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was decreased by all treatment methods, in which a small reduction was observed after

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treatment with UV and UV/Cl2, but a higher reduction was observed after the chlorination

307

treatment.

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Estimation of the effects on genome replication ability and the contribution of each

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vital function to MS2 inactivation. The genome reduction rate constant in each assay was 13 ACS Paragon Plus Environment


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compared with the inactivation rate constant to measure the proportion of each inactivation

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mechanism contributing to MS2 inactivation. In doing so, it was assumed that the total

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number of lost genome copies contributed to the loss of infectivity (kinfectivity) accounted for

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the number of genome copies lost due to an interruption of attachment (k'attachment), genome

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penetration (k'penetration), replication and post-replication processes (k'replication). The k'replication

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can be indirectly determined from the genome reduction rate due to damage to the viral

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genome, which was partially examined in this study (kobserved_damage) and could be estimated

317

(kestimated_damage) from the observed data obtained in this study. This concept was based on the

318

modified concept of a previous study24 and the details of the calculation are presented in the

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supporting information (Text S6). Accordingly, we could evaluate portions of k'replication that

320

contributed to MS2 inactivation in response to each treatment process (Table S5).

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Effects of UV254. Figure 6 shows that there was no contribution of the loss of attachment

322

ability to MS2 inactivation, whereas the loss of genome penetration ability was responsible

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for 14% of MS2 inactivation by the UV treatment, indicating that damage to important

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proteins employed during the genome penetration process occurred. A protein component

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called “A-protein” has been known to play an important role in promoting the attachment and

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genome penetration processes of bacteriophages35; and this result suggests that the A-protein

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was still functioning in the attachment process, however it malfunctioned during the genome

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penetration process after UV exposure. The genome penetration process has been shown to

329

be more complicated than the attachment process because RNA phages insert their genome

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into host cells as an A-protein RNA complex, rather than free RNA36, therefore, denaturation

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of the A-protein could inhibit the genome penetration process. Because proteins can absorb

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UV at 254 nm37, protein denaturation could be expected following UV irradiation, which has

333

been demonstrated by previous studies where UV induced damage to protein components of

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the viral capsid was observed using molecular techniques38–39. 14 ACS Paragon Plus Environment

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The estimated contribution of the loss of genome replication ability induced by UV was

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86%, which agreed with a number of recent studies showing an interruption of genome

337

replication in response to viral genome deterioration32, 40–41. It is well known that UV induces

338

random thymine-dimers on viral genomes. The RT-qPCR assay is a very sensitive method

339

that depends on the location and size of designed oligonucleotide sequences, but genome

340

damage may not be detected if a short length of the target genome is used, as shown in this

341

study. Another technique, such as long-range PCR, would be more appropriate to assess UV-

342

induced genome damage at other positions42. The results from this study suggest that the

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primary target of UV was the viral genome which can result in a failure to replicate the

344

genome. In contrast, the viral capsid is a less affected target.

345

Effects of Chlorine. Following the chlorination treatment, loss of replication ability

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amounted to 24% of the MS2 inactivation (Figure 6); the chlorine-induced genome damage

347

resulting in the loss of replication ability is different from UV-induced damage, with the viral

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genome being damaged by oxidation instead of dimer formation. The magnitude of damage

349

depends on the nucleobase contents with high guanine and cytosine contents providing an

350

opportunity for greater damage to the viral genome43. The predicted percent loss of genome

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replication ability following MS2 inactivation (42%), implied that greater damage occurred at

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positions of the viral genome other than the target region because the other regions may

353

contain higher guanine and cytosine contents. A new RT-qPCR technique using longer

354

sequence might be necessary for further studies to better understand the effects of chlorine on

355

the viral genome. A greater percent loss of the genome penetration ability was observed in

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response to the chlorination treatment (34%) than the UV treatment, which indicates a severe

357

impairment of the A-protein. Because chlorine has been shown to be able to alter the

358

conformation of proteins required for the genome penetration process44, the chlorine-induced

359

impairment of the A-protein observed in this study was expected. In conclusion, these results 15 ACS Paragon Plus Environment


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suggest that the viral genome was a primary target of the chlorination treatment, similar to the

361

UV treatment, but the effects on the viral capsid were greater than for UV.

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Effects of UV/Cl2. The attachment ability was not inhibited after UV/Cl2 treatment, which

363

indicates that exposure to OH• radicals did not decrease the function required for the

364

attachment of the A-protein. However, a loss of replication ability was observed as with the

365

chlorine treatment. Although, synergistic effects on genome damage were noted (2.1 times),

366

the contribution of the observed loss of replication ability to MS2 inactivation was only 17%.

367

The loss of genome penetration ability was only responsible for 6% of MS2 inactivation,

368

which was lower than either UV or chlorination. As we elucidated the effectiveness of

369

inhibiting effects on genome penetration by chlorine alone in a previous section, it is implied

370

that targets of chlorine and UV/Cl2 may be different and may be due to the OH• radicals.

371

Despite this, OH• radicals were found to not damage viral genome in this study, therefore it

372

likely causes damage to several locations of the viral capsid45 because of its non-specific

373

mode of action. As a result capsid cleavages can occur, and may facilitate chlorine diffusion

374

through these openings to harm viral genome directly. To verify the hypothesis, we

375

investigated genome sensitivity of MS2 in response to chlorine with different conditions of

376

MS2; namely intact MS2, capsid-damaged MS2 (details shown in Text S1), and naked MS2

377

RNA. It was found that naked MS2 RNA was the most sensitive to chlorine followed by

378

RNA extracted from capsid-damaged MS2 and intact MS2, respectively as shown in Figure

379

7. This supported that the chlorine can easily diffuse into OH•-induced capsid-damaged virus

380

than the intact virus and enhanced damages to viral genome. Moreover, the present of

381

chlorine species inside the capsid may induce formation of many active radicals not only OH•

382

to attack viral genome because of the change of pH. Namely, chlorine radicals (Cl•) and other

383

secondary radicals (Cl2•–, ClO•, ClOH•– and ClO•), a result of chain reactions between Cl• or

384

OH• and others46 may also contribute to the genome damage in the UV/Cl2 treatment. 16 ACS Paragon Plus Environment

Page 16 of 31

Page 17 of 31


385

Accordingly, the viral genome reduction rate in the UV/Cl2 was higher compared with either

386

UV or chlorine treatments alone observed in this study. This was consistent with the

387

estimated contribution of the loss of genome replication (77%), suggesting severe damage to

388

the viral genome on other positions. Based on these results, the OH• radicals and chlorine

389

seem to work in combination in the UV/Cl2 treatment and predominantly damaged viral

390

genome rather than A-protein to cause MS2 inactivation.

391

To our knowledge, this is the first study showing the presence of OH• radicals following

392

UV/Cl2 treatment with a practical chlorine dose (1 mg/L of free chlorine), and UV fluences

393

used for water disinfection; in addition this study is the first to show synergistic effects on

394

viral genome damage, which partially contributed to synergistic virus inactivation. Although

395

a model virus was used instead of enteric viruses, developing an understanding of how the

396

model virus is effectively inactivated by the UV/Cl2 treatment is important because it can be

397

used to predict how enteric viruses respond to UV/Cl2 treatment, but still investigation of

398

inactivation mechanisms with enteric viruses is needed for further studies. Based on the

399

fundamental knowledge obtained in this study, this effective inactivation can ensure that the

400

use of the simultaneous application of UV and chlorine in water treatment plants can enhance

401

the microbial safety of drinking water, which could lessen the concern regarding the use of

402

high UV fluences required for virus inactivation.

403

404

ACKNOWLEDGMENTS

405

Authors would like to express their deep appreciation to Prof. Madjid Mohseni, University of

406

British Columbia, for aiding us in measuring the radicals. This research was supported by the

407

Japan Science and Technology Agency (JST) and the Natural Science and Engineering

408

Research Council of Canada (NSERC) for the Japan-Canada Research Collaboration. The



409

Japan Society for the Promotion of Science (JSPS) also supported this study with Grants-in-

410

Aid for Scientific Research (26289181).

411 412

Supporting Information

413

The model used for the calculation of theoretical concentrations of the hydroxyl radicals;

414

estimates of the loss of genome replication ability; primers and probes used for RT-qPCR;

415

recovery rate in the attachment assay; standard curve generated by RT-qPCR; summary of the

416

reduction rate constants; preliminary results of the attachment assay with specific and non–

417

specific host cells; degradation of pCBA by UV photolysis; genome reduction and

418

inactivation profile of MS2 in response to UV/H2O2 treatment and genome reduction rate in

419

UV and chlorine measured by RT-qPCR and the attachment assay. This material is available

420

free of charge via the Internet at http://pubs.acs.org.

421 422

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FIGURE CAPTIONS

556 557

Figure 1. Illustrative schematic of experimental assays and purposes

558

Figure 2. Comparison between observed and predicted pCBA degradation in response to the

559

UV/Cl2 treatment, with an initial free chorine concentration of 1 mg/L.

560

Figure 3. Estimated inactivation profile of bacteriophage MS2 following exposure to 2.70 ×

561

10‒14 M OH• radicals from 3 independent experiments. The dashed lines show the 95%

562

confidence interval.

563

Figure 4. Genome reduction profile of bacteriophage MS2 in response to UV/Cl2 compared

564

with either UV or chlorination alone. The inactivation profile of UV+Cl2 was calculated.

565

Figure 5. Comparison of the genome reduction profiles of MS2 inactivated by UV, chlorine

566

and UV/Cl2 that were obtained using the RT-qPCR, attachment and genome penetration

567

assays.

568

Figure 6. Theoretical contribution of the loss of each vital function to the loss of infectivity

569

following treatment with UV, chlorine and UV/Cl2. The calculation was based on genome

570

reduction rate constant obtained from each assay in this study.

571

Figure 7. Genome sensitivity of intact MS2, capsid-damaged MS2 and naked MS2 RNA in

572

response to chlorine.

573 574 575 576 577 578 579


Page 24 of 31

Page 25 of 31


580 581

Figure 1. Illustrative schematic of experimental assays and purposes

582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600



601 602

Figure 2. Comparison between observed and predicted pCBA degradation in response to the

603

UV/Cl2 treatment, with an initial free chorine concentration of 1 mg/L.

604 605 606 607 608 609 610 611


Page 26 of 31

Page 27 of 31


612 613

Figure 3. Estimated inactivation profile of bacteriophage MS2 following exposure to 2.70 ×

614

10‒14 M OH• radicals from 3 independent experiments. The dashed lines show the 95%

615

confidence interval.

616 617 618 619 620 621 622 623 624 625 626 627 628 629 630



631 632

Figure 4. Genome reduction profile of bacteriophage MS2 in response to UV/Cl2 compared

633

with either UV or chlorination alone. The inactivation profile of UV+Cl2 was calculated.

634 635 636 637 638 639 640 641 642 643 644 645 646 647 648


Page 28 of 31

Page 29 of 31


649 650

Figure 5. Comparison of the genome reduction profiles of MS2 inactivated by UV, chlorine

651

and UV/Cl2 that were obtained using the RT-qPCR, attachment and genome penetration

652

assays. 29 ACS Paragon Plus Environment


653 654

Figure 6. Theoretical contribution of the loss of each vital function to the loss of infectivity

655

following treatment with UV, chlorine and UV/Cl2. The calculation was based on genome

656

reduction rate constant obtained from each assay in this study.

657 658 659 660 661 662 663 664 665 666 667 668 669 670


Page 30 of 31

Page 31 of 31


671 672

Figure 7. Genome sensitivity of intact MS2, capsid-damaged MS2 and naked MS2 RNA in

673

response to chlorine.


Analysis of Hydroxyl Radicals and Inactivation Mechanisms of

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