- Email: [email protected]
Norovirus and MS2 inactivation kinetics of UV-A and UV-B with and without TiO2
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
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/watres
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
Until recently, most UV disinfection studies have focused on germicidal 254 nm UV-C irradiation because of its high energy 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
mutagenic components of sunlight (Schiave et al. 2009). The 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 penetrating to the surface of Earth (Love et al. 2010). Solar irradiation 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 identified as the single most important factor in inactivation of
* Corresponding author. Tel.: þ82 2 880 2731; fax: þ82 2 745 9104. E-mail address: [email protected] (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 gastroenteritis, 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 calicivirus (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 inactivated by natural sunlight or pulsed UV light (Harding and Schwab, 2012; Jean et al. 2011). The efficacy of UV-C for inactivating various viruses, including NoVs, has been well characterized (Lee et al. 2008). However, the enteric virus inactivation kinetics of UV of other wavelengths, such as UVA 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 ultrafiltration (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 Dulbecco’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 Instrument Co., IL, USA). The incident light intensities from the UVA 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 suspension were 105e106 and 106e108 PFU/ml, respectively. MNV inactivation in TiO2 particles without UV appeared to be negligible in our experimental conditions. During the experiment, 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 experiments; and 0, 10, 20, and 30 min in MNV UV-B experiments), 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 instructions. 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. Realtime 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 inactivation 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 SigmaPlot (version 9.0, Systat Software, Inc.). SPSS (ver. 19.0; Armonk, NY, USA) was used for performing the KruskaleWallis test.
3.
Fig. 1 e Inactivation of tested viruses suspended in PBS by UV-A with and without TiO2 (n [ 3). (A) MS2 analyzed by SAL. (B) MNV by plaque assay. (C) MNV assayed by realtime RT-PCR.
Results
3.1. Inactivation of MS2 and MNV by UV-A and TiO2/UV-A Fig. 1 depicts the inactivation of MS2 and MNV by UV-A irradiation with and without TiO2. As shown in Fig. 1A, UV-A
irradiation displayed a constant, negligible effect on MS2 inactivation. The addition of TiO2 particles can enhance the inactivation efficacy of UV-A. An UV-A dose of 816 mJ/cm2 resulted in a 4 log10 reduction in MS2. A similar trend was 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 concentration 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 inactivation rates of TiO2 combined with UV-B were not significantly 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 inactivation 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 groundwater. 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 environmental conditions are shown in Table 1.
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
Fig. 2 e Inactivation of tested viruses suspended in PBS by UV-B with and without TiO2 (n [ 3). (A) MS2 analyzed by SAL. (B) MNV by plaque assay. (C) MNV assayed by realtime 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 efficiency 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 preexposure 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 photocatalytic reaction of TiO2 might occur through generation of O 2 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 markedly higher disinfection rate of UV-B compared with TiO2mediated inactivation, 2) limited capability for TiO2-mediated ROS generation by UV-B, or (3) blocking of UV-B by TiO2 particles. 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 inactivation (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 infectious virus particles. These results are consistent with a previous report (de Abreu Correˆa 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 region 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 mechanisms 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 reductions 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 UV-A with TiO2 Only UV-A UV-A with TiO2 Only UV-A UV-A with TiO2 Only UV-B UV-B with TiO2 Only UV-B UV-B with TiO2 Only UV-B UV-B with TiO2
0.0011 0.0049 0.0003 0.0029 0.00004 0.0028 0.0044 0.0057 0.0109 0.0123 0.0147 0.0123
0.0002 0.0001 0.00009 0.0006 0.00002 0.0002 0.0002 0.001 0.0017 0.002 0.0007 0.001
0.9798 0.9994 0.8376 0.9240 0.7959 0.9935 0.9978 0.9699 0.9847 0.9785 0.9986 0.9957
PBS PBS PBS PBS GW GW PBS PBS PBS PBS GW GW
MNV MNV UV-B
MS2 MNV MNV
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 Correˆa 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 presence of TiO2 is required to achieve significant MNV inactivation. 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 technologies are necessary for the effective control of viral pathogens.
Acknowledgments This research was supported by National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (grant number 2012-0009628, 2012-0008692).
references
Abbaszadegan, M., Huber, M.S., Gerba, C.P., Pepper, I.L., 1993. Detection of enteroviruses in groundwater with the polymerase chain reaction. Applied and Environmental Microbiology 59 (5), 1318e1324. Belliot, G., Lavaux, A., Souihel, D., Agnello, D., Pothier, P., 2008. Use of murine norovirus as a surrogate to evaluate resistance of human norovirus to disinfectants. Applied and Environmental Microbiology 74 (10), 3315e3318. Buckow, R., Isbarn, S., Knorr, D., Heinz, V., Lehmacher, A., 2008. Predictive model for inactivation of feline calicivirus, a norovirus surrogate, by heat and high hydrostatic
pressure? Applied and Environmental Microbiology 74 (4), 1030e1038. CDC, 2013. Surveillance for foodborne disease outbreaks e United States, 2009e2010. Morbidity and Mortality Weekly Report (MMWR) 62 (3), 41e47. Cho, M., Chung, H., Choi, W., Yoon, J., 2005. Different inactivation behaviors of MS-2 phage and Escherichia coli in TiO2 photocatalytic disinfection. Applied and Environmental Microbiology 71 (1), 270e275. Davies, C., Roser, D., Feitz, A., Ashbolt, N., 2009. Solar radiation disinfection of drinking water at temperate latitudes: inactivation rates for an optimised reactor configuration. Water Research 43 (3), 643e652. de Abreu Correˆa, A., Carratala, A., Barardi, C., Calvo, M., Girones, R., Bofill-Mas, S., 2012. Comparative inactivation of murine norovirus, human adenovirus, and human JC polyomavirus by chlorine in seawater. Applied and Environmental Microbiology 78 (18), 6450e6457. Doultree, J.C., Druce, J.D., Birch, C.J., Bowden, D.S., Marshall, J.A., 1999. Inactivation of feline calicivirus, a Norwalk virus surrogate. Journal of Hospital Infection 41 (1), 51e57. Duizer, E., Bijkerk, P., Rockx, B., De Groot, A., Twisk, F., Koopmans, M., 2004. Inactivation of caliciviruses. Applied and Environmental Microbiology 70 (8), 4538e4543. Girones, R., Ferru´s, M.A., Alonso, J.L., Rodriguez-Manzano, J., Calgua, B., de Abreu Correˆa, A., Hundesa, A., Carratala, A., Bofill-Mas, S., 2010. Molecular detection of pathogens in waterethe pros and cons of molecular techniques. Water Research 44 (15), 4325e4339. Godoy, P., Nuin, C., Alseda, M., Llovet, T., Mazana, R., Dominguez, A., 2006. Waterborne outbreak of gastroenteritis caused by Norovirus transmitted through drinking water. Revista Clinica Espanola 206 (9), 435e437. Hall, A.J., Eisenbart, V.G., Etingu¨e, A.L., Gould, L.H., Lopman, B.A., Parashar, U.D., 2012. Epidemiology of foodborne norovirus outbreaks, United States, 2001e2008. Emerging Infectious Diseases 18 (10), 1566e1573. Harding, A., Schwab, K., 2012. Using limes and synthetic psoralens to enhance solar disinfection of water (SODIS): a laboratory evaluation with norovirus, Escherichia coli, and MS2. The American Journal of Tropical Medicine and Hygiene 86 (4), 566e572. Hewitt, J., Rivera-Aban, M., Greening, G., 2009. Evaluation of murine norovirus as a surrogate for human norovirus and hepatitis A virus in heat inactivation studies. Journal of Applied Microbiology 107 (1), 65e71. Hollosy, F., 2002. Effects of ultraviolet radiation on plant cells. Micron 33 (2), 179e197.
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
Jean, J., Morales-Rayas, R., Anoman, M.-N., Lamhoujeb, S., 2011. Inactivation of hepatitis A virus and norovirus surrogate in suspension and on food-contact surfaces using pulsed UV light (pulsed light inactivation of food-borne viruses). Food Microbiology 28 (3), 568e572. King, B., Hoefel, D., Daminato, D., Fanok, S., Monis, P., 2008. Solar UV reduces Cryptosporidium parvum oocyst infectivity in environmental waters. Journal of Applied Microbiology 104 (5), 1311e1323. Ko, G., Cromeans, T.L., Sobsey, M.D., 2005. UV inactivation of adenovirus type 41 measured by cell culture mRNA RT-PCR. Water Research 39 (15), 3643e3649. Lee, J., Zoh, K., Ko, G., 2008. Inactivation and UV disinfection of murine norovirus with TiO2 under various environmental conditions. Applied and Environmental Microbiology 74 (7), 2111e2117. Leon, J., Lyon, G., Abdulhafid, G., Dowd, M., Hsiao, H., Liu, P., Wrammert, J., Ahmed, R., Schwab, K., Moe, C., 2007. Norovirus Human Infectivity Persists in Groundwater for Over Two Months. Abstr. 26th Am. Soc. Virol., Abstr. W, 9-1. Lim, M.Y., Kim, J.M., Lee, J.E., Ko, G., 2010. Characterization of ozone disinfection of murine norovirus. Applied and Environmental Microbiology 76 (4), 1120e1124. Love, D., Silverman, A., Nelson, K., 2010. Human virus and bacteriophage inactivation in clear water by simulated sunlight compared to bacteriophage inactivation at a Southern California beach. Environmental Science and Technology 44 (18), 6965e6970. Magulski, T., Paulmann, D., Bischoff, B., Becker, B., Steinmann, E., Steinmann, J., Goroncy-Bermes, P., 2009. Inactivation of murine norovirus by chemical biocides on stainless steel. BMC Infectious Diseases 9, 107e113. Maness, P.C., Smolinski, S., Blake, D.M., Huang, Z., Wolfrum, E.J., Jacoby, W.A., 1999. Bactericidal activity of photocatalytic TiO2 reaction: toward an understanding of its killing mechanism. Applied and Environmental Microbiology 65 (9), 4094e4098. Misstear, D., Gill, L., 2012. The inactivation of phages MS2, FX174 and PR772 using UV and solar photocatalysis. Journal of Photochemistry and Photobiology B: Biology 107, 1e8. Nuanualsuwan, S., Cliver, D., 2003. Capsid functions of inactivated human picornaviruses and feline calicivirus. Applied and Environmental Microbiology 69 (1), 350e357. Park, G., Boston, D., Kase, J., Sampson, M., Sobsey, M., 2007. Evaluation of liquid-and fog-based application of Sterilox hypochlorous acid solution for surface inactivation of human norovirus. Applied and Environmental Microbiology 73 (14), 4463e4468.
5613
Ryu, H., Gerrity, D., Crittenden, J.C., Abbaszadegan, M., 2008. Photocatalytic inactivation of Cryptosporidium parvum with TiO2 and low-pressure ultraviolet irradiation. Water Research 42 (6e7), 1523e1530. Sang, X., Phan, T., Sugihara, S., Yagyu, F., Okitsu, S., Maneekarn, N., Muller, W., Ushijima, H., 2007. Photocatalytic inactivation of diarrheal viruses by visible-light-catalytic titanium dioxide. Clinical Laboratory 53 (7e8), 413e421. Schiave, L.A., Pedroso, R.S., Candido, R.C., Roberts, D.W., Braga, G.U., 2009. Variability in UVB tolerances of melanized and nonmelanized cells of Cryptococcus neoformans and C. laurentii. Photochemistry and Photobiology 85 (1), 205e213. Seo, K., Lee, J.E., Lim, M.Y., Ko, G., 2012. Effect of temperature, pH, and NaCl on the inactivation kinetics of murine norovirus. Journal of Food Protection 75 (3), 533e540. Simonet, J., Gantzer, C., 2006. Inactivation of poliovirus 1 and Fspecific RNA phages and degradation of their genomes by UV irradiation at 254 nanometers. Applied and Environmental Microbiology 72 (12), 7671e7677. Sjogren, J., Sierka, R., 1994. Inactivation of phage MS2 by ironaided titanium dioxide photocatalysis. Applied and Environmental Microbiology 60 (1), 344e347. Thurston-Enriquez, J.A., Haas, C.N., Jacangelo, J., Riley, K., Gerba, C.P., 2003. Inactivation of feline calicivirus and adenovirus type 40 by UV radiation. Applied and Environmental Microbiology 69 (1), 577e582. USEPA, 2001. Methods 1602. Male-specific (Fþ) and Somatic Coliphage in Water by Single Agar Layer (SAL) Procedure. EPA Office of Water, Washington, DC, USA. Wegelin, M., Canonica, S., Mechsner, K., Fleischmann, T., Pesaro, F., Metzler, A., 1994. Solar water disinfection: scope of the process and analysis of radiation experiments. Journal of Water Supply: Research and Technology-Aqua 43 (4), 154e169. Widdowson, M.A., Sulka, A., Bulens, S.N., Beard, R.S., Chaves, S.S., Hammond, R., Salehi, E.D., Swanson, E., Totaro, J., Woron, R., Mead, P.S., Bresee, J.S., Monroe, S.S., Glass, R.I., 2005. Norovirus and foodborne disease, United States, 1991e2000. Emerging Infectious Diseases 11 (1), 95e102. Wigginton, K.R., Pecson, B.M., Sigstam, T.r., Bosshard, F., Kohn, T., 2012. Virus inactivation mechanisms: impact of disinfectants on virus function and structural integrity. Environmental Science and Technology 46 (21), 12069e12078. Wobus, C.E., Karst, S.M., Thackray, L.B., Chang, K.O., Sosnovtsev, S.V., Belliot, G., Krug, A., Mackenzie, J.M., Green, K.Y., Virgin, H.W., 2004. Replication of Norovirus in cell culture reveals a tropism for dendritic cells and macrophages. PLoS Biology 2 (12), 2076e2084.
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/watres
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
Until recently, most UV disinfection studies have focused on germicidal 254 nm UV-C irradiation because of its high energy 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
mutagenic components of sunlight (Schiave et al. 2009). The 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 penetrating to the surface of Earth (Love et al. 2010). Solar irradiation 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 identified as the single most important factor in inactivation of
* Corresponding author. Tel.: þ82 2 880 2731; fax: þ82 2 745 9104. E-mail address: [email protected] (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 gastroenteritis, 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 calicivirus (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 inactivated by natural sunlight or pulsed UV light (Harding and Schwab, 2012; Jean et al. 2011). The efficacy of UV-C for inactivating various viruses, including NoVs, has been well characterized (Lee et al. 2008). However, the enteric virus inactivation kinetics of UV of other wavelengths, such as UVA 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 ultrafiltration (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 Dulbecco’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 Instrument Co., IL, USA). The incident light intensities from the UVA 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 suspension were 105e106 and 106e108 PFU/ml, respectively. MNV inactivation in TiO2 particles without UV appeared to be negligible in our experimental conditions. During the experiment, 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 experiments; and 0, 10, 20, and 30 min in MNV UV-B experiments), 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 instructions. 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. Realtime 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 inactivation 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 SigmaPlot (version 9.0, Systat Software, Inc.). SPSS (ver. 19.0; Armonk, NY, USA) was used for performing the KruskaleWallis test.
3.
Fig. 1 e Inactivation of tested viruses suspended in PBS by UV-A with and without TiO2 (n [ 3). (A) MS2 analyzed by SAL. (B) MNV by plaque assay. (C) MNV assayed by realtime RT-PCR.
Results
3.1. Inactivation of MS2 and MNV by UV-A and TiO2/UV-A Fig. 1 depicts the inactivation of MS2 and MNV by UV-A irradiation with and without TiO2. As shown in Fig. 1A, UV-A
irradiation displayed a constant, negligible effect on MS2 inactivation. The addition of TiO2 particles can enhance the inactivation efficacy of UV-A. An UV-A dose of 816 mJ/cm2 resulted in a 4 log10 reduction in MS2. A similar trend was 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 concentration 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 inactivation rates of TiO2 combined with UV-B were not significantly 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 inactivation 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 groundwater. 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 environmental conditions are shown in Table 1.
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
Fig. 2 e Inactivation of tested viruses suspended in PBS by UV-B with and without TiO2 (n [ 3). (A) MS2 analyzed by SAL. (B) MNV by plaque assay. (C) MNV assayed by realtime 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 efficiency 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 preexposure 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 photocatalytic reaction of TiO2 might occur through generation of O 2 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 markedly higher disinfection rate of UV-B compared with TiO2mediated inactivation, 2) limited capability for TiO2-mediated ROS generation by UV-B, or (3) blocking of UV-B by TiO2 particles. 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 inactivation (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 infectious virus particles. These results are consistent with a previous report (de Abreu Correˆa 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 region 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 mechanisms 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 reductions 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 UV-A with TiO2 Only UV-A UV-A with TiO2 Only UV-A UV-A with TiO2 Only UV-B UV-B with TiO2 Only UV-B UV-B with TiO2 Only UV-B UV-B with TiO2
0.0011 0.0049 0.0003 0.0029 0.00004 0.0028 0.0044 0.0057 0.0109 0.0123 0.0147 0.0123
0.0002 0.0001 0.00009 0.0006 0.00002 0.0002 0.0002 0.001 0.0017 0.002 0.0007 0.001
0.9798 0.9994 0.8376 0.9240 0.7959 0.9935 0.9978 0.9699 0.9847 0.9785 0.9986 0.9957
PBS PBS PBS PBS GW GW PBS PBS PBS PBS GW GW
MNV MNV UV-B
MS2 MNV MNV
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 Correˆa 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 presence of TiO2 is required to achieve significant MNV inactivation. 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 technologies are necessary for the effective control of viral pathogens.
Acknowledgments This research was supported by National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (grant number 2012-0009628, 2012-0008692).
references
Abbaszadegan, M., Huber, M.S., Gerba, C.P., Pepper, I.L., 1993. Detection of enteroviruses in groundwater with the polymerase chain reaction. Applied and Environmental Microbiology 59 (5), 1318e1324. Belliot, G., Lavaux, A., Souihel, D., Agnello, D., Pothier, P., 2008. Use of murine norovirus as a surrogate to evaluate resistance of human norovirus to disinfectants. Applied and Environmental Microbiology 74 (10), 3315e3318. Buckow, R., Isbarn, S., Knorr, D., Heinz, V., Lehmacher, A., 2008. Predictive model for inactivation of feline calicivirus, a norovirus surrogate, by heat and high hydrostatic
pressure? Applied and Environmental Microbiology 74 (4), 1030e1038. CDC, 2013. Surveillance for foodborne disease outbreaks e United States, 2009e2010. Morbidity and Mortality Weekly Report (MMWR) 62 (3), 41e47. Cho, M., Chung, H., Choi, W., Yoon, J., 2005. Different inactivation behaviors of MS-2 phage and Escherichia coli in TiO2 photocatalytic disinfection. Applied and Environmental Microbiology 71 (1), 270e275. Davies, C., Roser, D., Feitz, A., Ashbolt, N., 2009. Solar radiation disinfection of drinking water at temperate latitudes: inactivation rates for an optimised reactor configuration. Water Research 43 (3), 643e652. de Abreu Correˆa, A., Carratala, A., Barardi, C., Calvo, M., Girones, R., Bofill-Mas, S., 2012. Comparative inactivation of murine norovirus, human adenovirus, and human JC polyomavirus by chlorine in seawater. Applied and Environmental Microbiology 78 (18), 6450e6457. Doultree, J.C., Druce, J.D., Birch, C.J., Bowden, D.S., Marshall, J.A., 1999. Inactivation of feline calicivirus, a Norwalk virus surrogate. Journal of Hospital Infection 41 (1), 51e57. Duizer, E., Bijkerk, P., Rockx, B., De Groot, A., Twisk, F., Koopmans, M., 2004. Inactivation of caliciviruses. Applied and Environmental Microbiology 70 (8), 4538e4543. Girones, R., Ferru´s, M.A., Alonso, J.L., Rodriguez-Manzano, J., Calgua, B., de Abreu Correˆa, A., Hundesa, A., Carratala, A., Bofill-Mas, S., 2010. Molecular detection of pathogens in waterethe pros and cons of molecular techniques. Water Research 44 (15), 4325e4339. Godoy, P., Nuin, C., Alseda, M., Llovet, T., Mazana, R., Dominguez, A., 2006. Waterborne outbreak of gastroenteritis caused by Norovirus transmitted through drinking water. Revista Clinica Espanola 206 (9), 435e437. Hall, A.J., Eisenbart, V.G., Etingu¨e, A.L., Gould, L.H., Lopman, B.A., Parashar, U.D., 2012. Epidemiology of foodborne norovirus outbreaks, United States, 2001e2008. Emerging Infectious Diseases 18 (10), 1566e1573. Harding, A., Schwab, K., 2012. Using limes and synthetic psoralens to enhance solar disinfection of water (SODIS): a laboratory evaluation with norovirus, Escherichia coli, and MS2. The American Journal of Tropical Medicine and Hygiene 86 (4), 566e572. Hewitt, J., Rivera-Aban, M., Greening, G., 2009. Evaluation of murine norovirus as a surrogate for human norovirus and hepatitis A virus in heat inactivation studies. Journal of Applied Microbiology 107 (1), 65e71. Hollosy, F., 2002. Effects of ultraviolet radiation on plant cells. Micron 33 (2), 179e197.
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
Jean, J., Morales-Rayas, R., Anoman, M.-N., Lamhoujeb, S., 2011. Inactivation of hepatitis A virus and norovirus surrogate in suspension and on food-contact surfaces using pulsed UV light (pulsed light inactivation of food-borne viruses). Food Microbiology 28 (3), 568e572. King, B., Hoefel, D., Daminato, D., Fanok, S., Monis, P., 2008. Solar UV reduces Cryptosporidium parvum oocyst infectivity in environmental waters. Journal of Applied Microbiology 104 (5), 1311e1323. Ko, G., Cromeans, T.L., Sobsey, M.D., 2005. UV inactivation of adenovirus type 41 measured by cell culture mRNA RT-PCR. Water Research 39 (15), 3643e3649. Lee, J., Zoh, K., Ko, G., 2008. Inactivation and UV disinfection of murine norovirus with TiO2 under various environmental conditions. Applied and Environmental Microbiology 74 (7), 2111e2117. Leon, J., Lyon, G., Abdulhafid, G., Dowd, M., Hsiao, H., Liu, P., Wrammert, J., Ahmed, R., Schwab, K., Moe, C., 2007. Norovirus Human Infectivity Persists in Groundwater for Over Two Months. Abstr. 26th Am. Soc. Virol., Abstr. W, 9-1. Lim, M.Y., Kim, J.M., Lee, J.E., Ko, G., 2010. Characterization of ozone disinfection of murine norovirus. Applied and Environmental Microbiology 76 (4), 1120e1124. Love, D., Silverman, A., Nelson, K., 2010. Human virus and bacteriophage inactivation in clear water by simulated sunlight compared to bacteriophage inactivation at a Southern California beach. Environmental Science and Technology 44 (18), 6965e6970. Magulski, T., Paulmann, D., Bischoff, B., Becker, B., Steinmann, E., Steinmann, J., Goroncy-Bermes, P., 2009. Inactivation of murine norovirus by chemical biocides on stainless steel. BMC Infectious Diseases 9, 107e113. Maness, P.C., Smolinski, S., Blake, D.M., Huang, Z., Wolfrum, E.J., Jacoby, W.A., 1999. Bactericidal activity of photocatalytic TiO2 reaction: toward an understanding of its killing mechanism. Applied and Environmental Microbiology 65 (9), 4094e4098. Misstear, D., Gill, L., 2012. The inactivation of phages MS2, FX174 and PR772 using UV and solar photocatalysis. Journal of Photochemistry and Photobiology B: Biology 107, 1e8. Nuanualsuwan, S., Cliver, D., 2003. Capsid functions of inactivated human picornaviruses and feline calicivirus. Applied and Environmental Microbiology 69 (1), 350e357. Park, G., Boston, D., Kase, J., Sampson, M., Sobsey, M., 2007. Evaluation of liquid-and fog-based application of Sterilox hypochlorous acid solution for surface inactivation of human norovirus. Applied and Environmental Microbiology 73 (14), 4463e4468.
5613
Ryu, H., Gerrity, D., Crittenden, J.C., Abbaszadegan, M., 2008. Photocatalytic inactivation of Cryptosporidium parvum with TiO2 and low-pressure ultraviolet irradiation. Water Research 42 (6e7), 1523e1530. Sang, X., Phan, T., Sugihara, S., Yagyu, F., Okitsu, S., Maneekarn, N., Muller, W., Ushijima, H., 2007. Photocatalytic inactivation of diarrheal viruses by visible-light-catalytic titanium dioxide. Clinical Laboratory 53 (7e8), 413e421. Schiave, L.A., Pedroso, R.S., Candido, R.C., Roberts, D.W., Braga, G.U., 2009. Variability in UVB tolerances of melanized and nonmelanized cells of Cryptococcus neoformans and C. laurentii. Photochemistry and Photobiology 85 (1), 205e213. Seo, K., Lee, J.E., Lim, M.Y., Ko, G., 2012. Effect of temperature, pH, and NaCl on the inactivation kinetics of murine norovirus. Journal of Food Protection 75 (3), 533e540. Simonet, J., Gantzer, C., 2006. Inactivation of poliovirus 1 and Fspecific RNA phages and degradation of their genomes by UV irradiation at 254 nanometers. Applied and Environmental Microbiology 72 (12), 7671e7677. Sjogren, J., Sierka, R., 1994. Inactivation of phage MS2 by ironaided titanium dioxide photocatalysis. Applied and Environmental Microbiology 60 (1), 344e347. Thurston-Enriquez, J.A., Haas, C.N., Jacangelo, J., Riley, K., Gerba, C.P., 2003. Inactivation of feline calicivirus and adenovirus type 40 by UV radiation. Applied and Environmental Microbiology 69 (1), 577e582. USEPA, 2001. Methods 1602. Male-specific (Fþ) and Somatic Coliphage in Water by Single Agar Layer (SAL) Procedure. EPA Office of Water, Washington, DC, USA. Wegelin, M., Canonica, S., Mechsner, K., Fleischmann, T., Pesaro, F., Metzler, A., 1994. Solar water disinfection: scope of the process and analysis of radiation experiments. Journal of Water Supply: Research and Technology-Aqua 43 (4), 154e169. Widdowson, M.A., Sulka, A., Bulens, S.N., Beard, R.S., Chaves, S.S., Hammond, R., Salehi, E.D., Swanson, E., Totaro, J., Woron, R., Mead, P.S., Bresee, J.S., Monroe, S.S., Glass, R.I., 2005. Norovirus and foodborne disease, United States, 1991e2000. Emerging Infectious Diseases 11 (1), 95e102. Wigginton, K.R., Pecson, B.M., Sigstam, T.r., Bosshard, F., Kohn, T., 2012. Virus inactivation mechanisms: impact of disinfectants on virus function and structural integrity. Environmental Science and Technology 46 (21), 12069e12078. Wobus, C.E., Karst, S.M., Thackray, L.B., Chang, K.O., Sosnovtsev, S.V., Belliot, G., Krug, A., Mackenzie, J.M., Green, K.Y., Virgin, H.W., 2004. Replication of Norovirus in cell culture reveals a tropism for dendritic cells and macrophages. PLoS Biology 2 (12), 2076e2084.