w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 5 6 0 7 e5 6 1 3

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Norovirus and MS2 inactivation kinetics of UV-A

and UV-B with and without TiO2

Jung Eun Lee a,b, GwangPyo Ko b,*

a
Han River Environment Research Center, National Institute of Environmental Research, 819 Yangsoo-ri,
Yangpyeong-goon, Gyeonggi Province 476-823, Republic of Korea
b
Department of Environmental Health and Institute of Health and Environment, School of Public Health,
Seoul National University, 1st Gwanak-ro, Gwanak-gu, Seoul 151-742, Republic of Korea

article info abstract

Article history: Germicidal ultraviolet, such as 254-nm UV-C, is a common method of disinfection of
Received 11 April 2013 pathogenic enteric viruses. However, the disinfection efficacies of UV-A or -B in terms of
Received in revised form inactivating waterborne viruses such as norovirus have not been characterized. We eval-
29 May 2013 uated the inactivation kinetics of MS2 bacteriophage and murine norovirus (MNV), a sur-
Accepted 18 June 2013 rogate of human norovirus (NoV), by UV-A and -B. In addition to UV disinfection, we
Available online 29 June 2013 further investigated whether the presence of TiO2 could enhance the virus inactivation
kinetics of UV-A and -B. Both MS2 and MNV were highly resistant to UV-A. However, the
Keywords: addition of TiO2 enhanced the efficacy of UV-A for inactivating these viruses. UV-A dose of
UV-A inactivation 1379 mJ/cm2 resulted in a 4 log10 reduction. In comparison, UV-B alone effectively inacti-
UV-B inactivation vated both MS2 and MNV, as evidenced by the 4 log10 reduction by 367 mJ/cm2 of UV-B. The
Norovirus addition of TiO2 increased the inactivation of MS2; however, it did not significantly increase
Murine norovirus the efficacy of UV-B disinfection for inactivating MNV. When these treatments were
MS2 phage applied to field water such as groundwater, the results were generally consistent with the
Titanium dioxide laboratory findings. Our results clearly indicated that UV-B is useful for the disinfection of
waterborne norovirus. However, MNV was quite resistant to UV-A, and UV-A effectively
inactivated the tested viruses only when used in combination with TiO2.
ª 2013 Elsevier Ltd. All rights reserved.

1. Introduction mutagenic components of sunlight (Schiave et al. 2009). The

Until recently, most UV disinfection studies have focused on
germicidal 254 nm UV-C irradiation because of its high en-
ergy and absorbance by nucleic acids, resulting in efficient
inactivation of various microorganisms (Ko et al. 2005).
However, UV light covers a wide range of wavelengths, and
those other than UV-C are also considered to be germicidal
(King et al. 2008). For example, traditionally, sunlight has
been considered to be germicidal. UV-B (280e320 nm) and
UV-A (320e400 nm) are the most biologically damaging and
UV fraction of the solar spectrum that reaches the surface of
the Earth is composed mainly of UV-A and UV-B radiation
because atmospheric ozone blocks most UV-C from pene-
trating to the surface of Earth (Love et al. 2010). Solar irra-
diation has been used as one of the main environmental
methods of reducing widespread risk to human health
from water-borne pathogenic microorganisms worldwide,
particularly in developing countries (Davies et al. 2009;
Schiave et al. 2009). Solar radiation has also been identi-
fied as the single most important factor in inactivation of

* Corresponding author. Tel.: þ82 2 880 2731; fax: þ82 2 745 9104.
E-mail address: gko@snu.ac.kr (GwangPyo Ko).
0043-1354/$ e see front matter ª 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.watres.2013.06.035
5608 w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 5 6 0 7 e5 6 1 3
allochthonous microorganisms in natural waters (Davies
et al. 2009).
Noroviruses (NoVs) are food- and waterborne viruses
responsible for more than 90% of acute nonbacterial gastroen-
teritis, and recently became epidemic worldwide (Godoy et al.
2006; Hall et al. 2012; Widdowson et al. 2005). According to an
American Center for Disease Control and Prevention report in
2009e2010, NoV was the most common cause of outbreak and
illness, accounting for 331 (42%) of confirmed single-etiology
outbreaks and 7332 (37%) illnesses (CDC, 2013). NoV is known
to be highly infectious and has a low infectious dose (w10 viral
particles) (Seo et al. 2012). The viral infectivity of NoVs persists for
over 2 months in groundwater (Leon et al. 2007). Hence, the
effective management and disinfection of NoVs in water is
important for public health. An assay of viral infectivity is critical
for determining the inactivation rates of NoV. However, no
conventional culture assay for human NoVs has yet been
developed (Doultree et al. 1999). Therefore, other biologically
similar viruses, such as murine norovirus (MNV) and feline cal-
icivirus (FCV), have been used as surrogates for human NoV
(Buckow et al. 2008; Wobus et al. 2004). Using these surrogate
viruses, a number of studies have reported inactivation of NoV by
chemical biocides (Magulski et al. 2009), heat (Hewitt et al. 2009),
ultraviolet (Lee et al. 2008) and other disinfectants (Belliot et al.
2008). Because bacteriophages such as MS2 are easy to grow,
fast-growing, and non-pathogenic, they have been widely used
as surrogates for viral pathogens in various disinfection settings
(Misstear and Gill, 2012; Wigginton et al. 2012). Therefore, MS2
could be applied as a viral surrogate for comparison with the
experimental setting and data in previous studies.
Recent studies have suggested that enteric viruses
including MNV and hepatitis A virus (HAV) could be inacti-
vated by natural sunlight or pulsed UV light (Harding and
Schwab, 2012; Jean et al. 2011). The efficacy of UV-C for inac-
tivating various viruses, including NoVs, has been well char-
acterized (Lee et al. 2008). However, the enteric virus
inactivation kinetics of UV of other wavelengths, such as UV-
A or -B, have not been well characterized. These data are
particularly important because many regions of the world use
solar irradiation as the primary disinfection method (King
et al. 2008). We also investigated the efficacy of inactivation
using titanium dioxide (TiO2), which is a photocatalyst that
could enhance inactivation by UV light. TiO2 may represent an
attractive alternative treatment of contaminated wastewater
or surface water, as well as facilitate the purification and
disinfection of drinking water (Misstear and Gill, 2012). The
objectives of this study were to characterize the inactivation
kinetics of viruses, including MNV and MS2, by UV-A and -B,
and to determine whether TiO2 can enhance the inactivation
rates of these viruses by UV-A and -B.
2. Materials and methods
2.1. Preparation of MS2 and MNV stocks
Bacteriophage MS2 (ATCC No. 15597-B1) was cultured and
analyzed by single agar layer (SAL) methods of the United
States Environmental Protection Agency (USEPA, 2001). To
prepare the MS2 stock, viruses were purified from infected cell
lysates of a single agar layer plaque assay plate with confluent
lysis by extraction with an equal volume of chloroform, and
centrifuged at 4000 g for 30 min. The supernatant was
recovered and stored at 80  C.
The MNV stock was prepared using RAW 264.7 cells and
plaque assay, as described previously (Lee et al. 2008). Briefly,
virus was cultured in RAW 264.8 cells with Dulbecco’s modified
Eagle’s medium (DMEM; Gibco, Grand Island, NY) containing
10% fetal bovine serum (Gibco). Viruses were inoculated and
cultivated on confluent RAW 264.7 cell monolayers for 3e4
days. Infected cells were subjected to freezing and thawing
three times to release the viruses. Cell debris was removed by
centrifugation at 2000 g for 10 min at 4  C. To further
concentrate the MNV, we subjected the supernatant to ultra-
filtration (Amicon Ultra-15; Millipore, Billerica, MA) at 5000 g
for 10 min at 4  C. The supernatant was recovered from the filter
unit and stored at 80  C until use. We performed a plaque
assay to enumerate infectious MNV. Briefly, RAW 264.7 cells
were seeded into 60-mm plates and allowed to adhere. Serial
dilutions of MNV were made on ice using supplemented Dul-
becco’s MEM. After decanting media from the plates, the cells
were inoculated with 0.5 ml of diluted virus suspensions. After
incubation for 1 h, the inocula were aspirated and replaced with
SeaPlaque agarose containing supplemented MEM, allowed to
solidify, and incubated until plaques became visible. Neutral
red solution was added for better visualization of the plaques.
2.2. Experimental design for disinfection using UV-A
and -B
A collimated beam UV apparatus containing two UV lamps
was used as described in previous studies (Ko et al. 2005) and
UV-A and -B lamps were used. The irradiance of both UV-A
one lamp (10 W, Sankyo Denki Co., Japan) and UV-B two
lamp (8 W, Sankyo Denki Co., Japan) was measured using a
VLX3W radiometer (CX-365 and CX-312, ColeeParmer Instru-
ment Co., IL, USA). The incident light intensities from the UV-
A and -B lamps on the reactor surface were measured
(0.14 mW/cm). Before the experiment, the lamp was warmed
up for 30 min and we measured the irradiance before and after
the experiment. TiO2 particle (Degussa Co., Incheon, Korea)
suspensions were prepared using 1 g/L PBS (Maness et al. 1999)
or filtered (0.22 mm) groundwater and sonicated for 30 min
before the experiment. The TiO2 suspension was pre-exposed
to UV light for 5 min with ROS to obtain a steady state and
increase the photocatalytic efficiency (Ryu et al. 2008). Viral
stocks were diluted 1/50 in PBS, and 100 ml of viral suspension
was placed in a Petri dish (60  15 mm) containing 10 ml of PBS
or TiO2 suspension. The viral titers of MNV and MS2 in sus-
pension were 105e106 and 106e108 PFU/ml, respectively. MNV
inactivation in TiO2 particles without UV appeared to be
negligible in our experimental conditions. During the experi-
ment, the suspension was mixed using a magnetic stirrer. At
pre-determined sampling times (0, 30, 60, 90, and 120 min in
MS2 UV-A experiments; 0, 30, 60, 120, 180, and 210 min MNV
UV-A experiments; 0, 20, 40, 60, and 80 min in MS2 UV-B ex-
periments; and 0, 10, 20, and 30 min in MNV UV-B experi-
ments), 0.5 ml of viral suspension was assayed by both the
plaque assay and real-time TaqMan RT-PCR. The experiments
were repeated three times. An experiment without UV
 w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 5 6 0 7 e5 6 1 3 5609

irradiation or TiO2 particles was performed as a control under

the same conditions. Groundwater was obtained from a rural
region in Gyeonggi Province. The turbidity of the groundwater
was 0.42 nephelometric turbidity units (NTU). The pH of
groundwater, measured prior to the experiment, was 7.25.

2.3. Nucleic acid extraction and quantitation of viral

nucleic acids

To extract viral RNA, we used the QIAamp Viral Mini Kit

(Qiagen, Valencia, CA) according to the manufacturer’s in-
structions. To remove the particles after TiO2 inactivation, the
sample suspension was centrifuged at 10,000 g for 1 min at
room temperature. Briefly, 140 ml of virus suspension was
mixed with 560 ml of guanidinium thiocyanate lysis solution,
followed by RNA precipitation with an equal volume of 96%e
100% ethanol. Viral RNA was further purified using a QIAamp
Mini Column (Qiagen Viral RNA Kit). Purified RNA samples
were stored at 80  C until RT-PCR analysis.
To quantify viral nucleic acids, we prepared the standard
using the capsid region and used a serial dilution for each
experiment. The forward primer (50 -ACG CCA CTC CGC ACA
AA-30 ), reverse primer (50 -GCG GCC AGA GAC CAC AAA-30 ),
and probe (VIC-AGC CCG GGT GAT GAG-MGB) were used for
real-time RT-PCR of MNV, as described in a previous study
(Lee et al. 2008). For quantitation of viral genes, serial dilution
of the capsid gene cloned in a TA cloning vector (Promega,
Madison, WI) was used to generate a standard curve. Real-
time RT-PCR was performed using an ABI 7300 real-time PCR
instrument (Applied Biosystems, Foster City, CA). The final
reaction volume of 25 ml contained 2.5 ml of MNV viral RNA,
primers (0.4 M each), fluorescently labeled probe (0.1 mM), plus
the nucleotides, RTng-PCR enzyme mix, and buffer provided
in the AgPath-ID One-step RT-PCR kit (Ambion, Austin, TX).
The reactions were performed at 42  C for 10 min and 95  C for
10 min, followed by 45 cycles of 95  C for 15 s and 60  C for 60 s.

2.4. Quantitative analysis of viral inactivation

Viral inactivation was assessed using Chick’s law, as described

previously (Thurston-Enriquez et al. 2003). Briefly, UV inacti-
vation can be calculated by the following equation: Nt/No ¼ e
ekit
, where Nt represents the number of viral particles at time t
(UV exposure duration, s), No is the number of viral particles at
time zero (no UV irradiation), K is the slope of the inactivation
curve, i is the intensity of UV light energy (mW/cm2). The
parameter log of the survival ratio (Nt/No) versus UV dose for
each experiment was used to perform regression analysis for
each water type and with/without TiO2 particles using Sigma-
Fig. 1 e Inactivation of tested viruses suspended in PBS by
Plot (version 9.0, Systat Software, Inc.). SPSS (ver. 19.0; Armonk,
UV-A with and without TiO2 (n [ 3). (A) MS2 analyzed by
NY, USA) was used for performing the KruskaleWallis test.
SAL. (B) MNV by plaque assay. (C) MNV assayed by real-
time RT-PCR.

3. Results

3.1. Inactivation of MS2 and MNV by UV-A and irradiation displayed a constant, negligible effect on MS2
TiO2/UV-A inactivation. The addition of TiO2 particles can enhance the
inactivation efficacy of UV-A. An UV-A dose of 816 mJ/cm2
Fig. 1 depicts the inactivation of MS2 and MNV by UV-A irra- resulted in a 4 log10 reduction in MS2. A similar trend was
diation with and without TiO2. As shown in Fig. 1A, UV-A observed for MNV inactivation (Fig. 1B). The concentrations of
5610 w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 5 6 0 7 e5 6 1 3

MS2 and MNV at the end of inactivation were 20e240 and

2e20 PFU/ml, respectively. However, a higher dose of UV-A in
the presence of TiO2 was required to inactivate MNV
compared to MS2. For MNV, an UV-A dose of 1379 mJ/cm2
resulted in a 4 log10 reduction. Regardless, the combined TiO2/
UV-A treatment showed remarkable rates of inactivation of
both MS2 and MNV. Finally, the MNV nucleic acid concen-
tration was consistent regardless of the degree of inactivation,
as measured by plaque assay. By real-time RT-PCR assay, no
significant reduction in MNV was observed (Fig. 1C).

3.2. Inactivation of MS2 and MNV by UV-B and

TiO2/UV-B

Fig. 2 shows MS2 and MNV inactivation by UV-B with and

without TiO2. As shown in Fig. 2A, a w4 log10 reduction in MS2
occurred upon exposure to the UV-B dose of 909 mJ/cm2. The
concentrations of MS2 at the end of inactivation were
102e104 PFU/ml. A significant increase in inactivation rates
was observed with the combined TiO2/UV-B treatment. A w4
log10 inactivation of MS2 was achieved with an UV-B dose of
702 mJ/cm2 in the presence of TiO2. Exposure to an UV-B dose
of 367 mJ/cm2 resulted in a 4 log10 reduction in MNV (Fig. 2B).
The concentrations of MNV at the end of inactivation were
24e800 PFU/ml. Unlike the previous results, the MNV inacti-
vation rates of TiO2 combined with UV-B were not signifi-
cantly different from those of UV-B alone. Overall, MNV was
significantly more susceptible to UV-B than MS2 (P < 0.05).
When assayed by real-time RT-PCR, no significant reduction
in MNV was detected, similar to the UV-A results (Fig. 2C).

3.3. Inactivation of MNV in groundwater by UV-A and

-B with and without TiO2

Inactivation curves of MNV by either UV-A or -B with and

without TiO2 are shown in Fig. 3. In groundwater, the inacti-
vation rates of MNV by UV-A or -B with and without TiO2 were
similar to those in PBS (P > 0.05), except for UV-B without TiO2
(P < 0.05). As shown in Fig. 3A, no reduction in MNV occurred
using UV-A alone. UV-A, at a dose of 1492 mJ/cm2 with TiO2,
was needed to achieve a 4 log10 reduction of MNV in ground-
water. Using UV-B without and with TiO2, UV-B doses of 272
and 325 mJ/cm2, respectively, were required to achieve a 4
log10 reduction in MNV (Fig. 3B). The concentrations of MNV at
the end of inactivation were 6e180 PFU/ml. The first-order
inactivation kinetics of the viruses under various environ-
mental conditions are shown in Table 1.

Fig. 2 e Inactivation of tested viruses suspended in PBS by

4. Discussion
In this study, we investigated the inactivation of MS2 and
MNV. Both MNV and MS2 were used as viral surrogates for
human NoVs. To our knowledge, this is the first report of MS2
and MNV inactivation by both UV-A and -B. In addition, we
evaluated whether TiO2 enhanced the efficacies of UV-A and
-B disinfection. Despite the significance of NoV disinfection in
public health, the efficiency of disinfection by non-UV-C with
and without TiO2 has not been described. Our data suggested
UV-B to be an effective method of NoV inactivation. With
UV-B with and without TiO2 (n [ 3). (A) MS2 analyzed by
SAL. (B) MNV by plaque assay. (C) MNV assayed by real-
time RT-PCR.
UV-A, NoV was significantly inactivated only in the presence
of TiO2.
When MS2 was exposed to UV-A, the virus was inactivated
only negligibly, which is consistent with a previous report
(Cho et al. 2005). Like MS2, MNV was not significantly
 w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 5 6 0 7 e5 6 1 3
Fig. 3 e Inactivation of MNV suspended in groundwater by
UV-A with and without TiO2 (n [ 3) (A) and UV-B with and
without TiO2 (n [ 3) (B).
inactivated. However, in the presence of TiO2, both viruses
were markedly inactivated by UV-A (Fig. 1A and B). The effi-
ciency of TiO2 in terms of inactivation of microorganisms has
been reported by others (Davies et al. 2009; Maness et al. 1999).
Our results also showed markedly increased inactivation of
both MS2 and MNV by UV-A with TiO2. One study reported
significant inactivation of MS2 by TiO2/UV-A (Sjogren and
Sierka, 1994); however, the MS2 inactivation rate in our
study was slightly higher than that of the previous study. One
reason for the apparent difference could be the effect of pre-
exposure to UV light. In our study, TiO2 was pre-exposed for
5 min in the absence of MS2. TiO2 pre-exposure allowed for
complete mixing of the TiO2 suspension, and TiO2/UV with
pre-exposure showed better inactivation than UV/TiO2 (Ryu
et al. 2008). Upon exposure to TiO2/UV-A (1034 mJ/cm2), the
MNV inactivation rates increased dramatically (>3 log10). MNV
was more resistant than MS2 to inactivation by TiO2/UV-A. A
previous study reported that virus inactivation by the photo-
catalytic reaction of TiO2 might occur through generation of
O2 and *OH followed by damage to the viral capsid protein and
genome (Sang et al. 2007). Another oxidation study of HOCl
5611
inactivation reported that MNV was more resistant to HOCl
than MS2 (Park et al. 2007).
Compared to UV-A, the disinfection efficacy of UV-B for a
wide range of organisms such as bacteria, fungi and plants is
better established (King et al. 2008). However, studies of UV-B
inactivation of viruses are limited. One previous study
assessed the inactivation of other animal caliciviruses (FeCV
and CaCV) using 280e320-nm UV-B and the TCID50 culture
method (Duizer et al. 2004). These caliciviruses were more
sensitive to UV-B than MNV. MS2 was significantly more
inactivated by UV-B in the presence of TiO2. In contrast, the
presence of TiO2 did not enhance the inactivation of MNV by
UV-B. Duizer et al. also reported that animal caliciviruses
(FeCV and CaCV) were more sensitive to UV-B than MS2 phage
(Duizer et al. 2004). These results may be due to 1) the mark-
edly higher disinfection rate of UV-B compared with TiO2-
mediated inactivation, 2) limited capability for TiO2-mediated
ROS generation by UV-B, or (3) blocking of UV-B by TiO2 par-
ticles. Further research is warranted to investigate the
possible mechanisms.
The major target of UV disinfection is the viral nucleic acid
(RNA) (Nuanualsuwan and Cliver, 2003). MS2 and MNV have
genomes of different sizes (3569 vs. 7382 bases). One previous
study reported that MS2 phage (3569 bases) was significantly
more resistant than poliovirus 1 (7440 bases) to UV inactiva-
tion (Simonet and Gantzer, 2006). This phenomenon is likely
related more to the difference in genome size than the capsid
structure (Simonet and Gantzer, 2006). UV-B can induce a
variety of damaging effects in microbes, and UV-A represents
the less hazardous portion of UV radiation (Hollosy, 2002).
The disinfection effect of UV-A is quite limited. Therefore, to
obtain significant inactivation, a longer exposure duration or
high UV irradiance is typically necessary (Wegelin et al. 1994).
UV-C is highly effective for inactivating norovirus (Lee et al.,
2008). In our study, viral plaque assay and real-time RT-PCR
were used to measure infectious viruses and viral nucleic
acid, respectively. The reduction in viral nucleic acid by UV-A
and -B is limited, despite the significant reduction in infec-
tious virus particles. These results are consistent with a pre-
vious report (de Abreu Corrêa et al., 2012; Girones et al., 2010;
Lee et al., 2008). UV is known to damage nucleic acids
(Wigginton et al. 2012). However, when viral nucleic acid was
quantified by real-time TaqMan RT-PCR assay, the target re-
gion was small (only 55 bases of the 7.6-kb viral genome), so
the other nucleic acid region was damaged but could still
be amplified by real-time RT-PCR assay. A previous
study indicated that inactivation rates measured by RT-PCR
were significantly affected by the template size of the
RT-PCR amplicon (Lim et al. 2010). Other possible mecha-
nisms could be UV-B damaging other cellular components,
such as proteins, due to its high energy and penetrating
characteristics.
The doses of UV-A and -B required to achieve 4 log10 re-
ductions were similar for both PBS and groundwater samples.
The higher turbidity and presence of other chemicals in the
groundwater did not significantly affect the susceptibility of
MNV to UV. These results are similar to those of a previous
study (Thurston-Enriquez et al. 2003) that reported similar
feline calicivirus inactivation rates by UV-C between BDF
and treated groundwater samples. The presence of other
5612 w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 5 6 0 7 e5 6 1 3

Table 1 e Summary of the means ± standard error of decay values (K ) and R2 values from UV disinfection experiments.
Ultraviolet Virus UV experiment K SE of K R2 Water typea

UV-A MS2 Only UV-A 0.0011 0.0002 0.9798 PBS

UV-A with TiO2 0.0049 0.0001 0.9994 PBS
MNV Only UV-A 0.0003 0.00009 0.8376 PBS
UV-A with TiO2 0.0029 0.0006 0.9240 PBS
MNV Only UV-A 0.00004 0.00002 0.7959 GW
UV-A with TiO2 0.0028 0.0002 0.9935 GW
UV-B MS2 Only UV-B 0.0044 0.0002 0.9978 PBS
UV-B with TiO2 0.0057 0.001 0.9699 PBS
MNV Only UV-B 0.0109 0.0017 0.9847 PBS
UV-B with TiO2 0.0123 0.002 0.9785 PBS
MNV Only UV-B 0.0147 0.0007 0.9986 GW
UV-B with TiO2 0.0123 0.001 0.9957 GW

a GW: filtered (0.22 mm) groundwater.

chemicals and humic acids could affect the viral recovery and
disinfection rates (Abbaszadegan et al. 1993). For example, a
previous study reported that natural organic compounds in
seawater might affect the inactivation rates of viruses (de
Abreu Corrêa et al. 2012). Detailed further studies of viral
inactivation by UV-A or -B under the conditions present in
natural water sources, such as pH, and heavy metal and
organic matter levels, should be performed.
5. Conclusion
The current study demonstrated the effectiveness of UV-B
disinfection for inactivation of MNV. With UV-A, the pres-
ence of TiO2 is required to achieve significant MNV inactiva-
tion. Our results indicated the potential of UV-A, UV-B, or solar
irradiation for disinfection of viral pathogens in water,
although careful consideration and a combination of tech-
nologies are necessary for the effective control of viral
pathogens.
Acknowledgments
This research was supported by National Research Founda-
tion of Korea (NRF) funded by the Ministry of Education,
Science and Technology (grant number 2012-0009628,
2012-0008692).
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