International Journal of Food Microbiology 211 (2015) 73–78

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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro

Short communication

Ultraviolet-C efficacy against a norovirus surrogate and hepatitis A virus on a stainless steel surface Shin Young Park, An-Na Kim, Ki-Hoon Lee, Sang-Do Ha ⁎ School of Food Science and Technology, Chung-Ang University, 72–1 Nae-Ri, Daeduck-Myun, Ansung, Kyunggido 456–756, Republic of Korea

a r t i c l e

i n f o

Article history: Received 9 December 2014 Received in revised form 1 July 2015 Accepted 5 July 2015 Available online 10 July 2015 Keywords: Murine norovirus-1 Hepatitis A virus Ultraviolet-C Stainless steel surface Modified Gompertz equation

a b s t r a c t In this study, the effects of 10–300 mWs/cm2 of ultraviolet radiation (UV-C) at 260 nm were investigated for the inactivation of two foodborne viruses: murine norovirus-1 (MNV-1; a human norovirus [NoV] surrogate) and hepatitis A virus (HAV). We used an experimentally contaminated stainless steel surface, a common foodcontact surface, to examine the effects of low doses of UV-C radiation on MNV-1 and HAV titers. The modified Gompertz equation was used to generate non-linear survival curves and calculate dR-values as the UV-C dose of 90% reduction for MNV-1 (R2 = 0.95, RMSE = 0.038) and HAV (R2 = 0.97, RMSE = 0.016). Total MNV-1 and HAV titers significantly decreased (p b 0.05) with higher doses of UV-C. MNV-1 and HAV were reduced to 0.0–4.4 and 0.0–2.6 log10PFU/ml, respectively, on the stainless steel surfaces by low-dose UV-C treatment. The dR-value, 33.3 mWs/cm2 for MNV-1 was significantly (p b 0.05) lower than 55.4 mWs/cm2 of HAV. Therefore, the present study shows that HAV is more resistant to UV-C radiation than MNV-1. These data suggest that low doses of UV-C light on food contact surfaces could be effective to inactivate human NoV and HAV in restaurant, institutional, and industrial kitchens and facilities. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Norovirus (NoV), hepatitis A virus (HAV), rotavirus, sapovirus, and astrovirus are all major pathogens that cause foodborne illnesses in the United States (Scallan et al., 2011). Among the enteric viruses, NoV is the leading cause of non-bacterial gastroenteritis and is associated with 80 to 90% of reported outbreaks in both developing and developed countries (Glass et al., 2009). HAV infections are also a leading cause of foodborne disease outbreaks occurring regularly in both developing and developed countries (Koopmans and Duizer, 2004; Richards, 2001). While NoV belongs to the Caliciviridae family and HAV belongs to the Picornaviridae family, both viruses are primarily transmitted via the fecal–oral route, either by direct person-to-person contact or ingestion of contaminated food and water. Together, NoV and HAV infections are considered to be the most common causes of non-bacterial gastroenteritis, a disease largely resulting from contaminated shellfish (Woods and Burkhardt, 2010; Wright et al., 2009), fresh produce (Butot et al., 2009; Donnan et al., 2012; Heaton and Jones, 2008; Lynch et al., 2009), and ready-to-eat food (Malek et al., 2009; Schmid et al., 2007) around the world. The transmission of pathogenic microorganisms to food via contaminated surfaces is a significant problem in food processing, catering, and domestic environments. Specifically, ⁎ Corresponding author at: Dept. of Food Science and Technology, Chung-Ang University, 72–1 Nae-ri, Daeduk-myun, Ansung, Gyunggido 456–756, Republic of Korea. E-mail address: [email protected] (S.-D. Ha).

http://dx.doi.org/10.1016/j.ijfoodmicro.2015.07.006 0168-1605/© 2015 Elsevier B.V. All rights reserved.

food-contact surfaces, which are typically made of stainless steel, polypropylene, glass, or wood are used in food processing plants and kitchens. These surfaces can be contaminated as a result of poor personal hygiene, leading to the transfer of the virus to various food products (D'Souza et al., 2006). Among these food-contact surfaces, stainless steel is widely used in food manufacturing and processing industries for manufacture, bulk storage and transportation, preparation, and presentation applications. The use of disinfectants is a key intervention measure to interrupt the transmission of pathogenic microorganisms, including NoV and HAV, onto food-contact surfaces. The U.S. Food and Drug Administration (FDA) has approved the use of UV-C on food products for controlling surface microorganisms (US FDA, 2007). UV-C radiation in the wavelength range 250–280 nm is normally used to disinfect water, cooking utensils, and liquid foods (Guerrero-Beltrán and Barbosa-Cánovas, 2004). This is especially ideal because UV-C produces no unpleasant odor and has a low energy requirement when applied to foods, utensils, and water. Moreover, UV-C radiation is known to possess antiviral activity in food and water (Fino and Kniel, 2008; Ko et al., 2005; Lenes et al., 2010; Nwachuku et al., 2005; Park et al., 2011) and food contact and swine farm surfaces (Dee et al., 2011; Li et al., 2011). After studying the effects of UV-C on feline calicivirus (FCV; a NoV surrogate) and HAV, Fino and Kniel (2008) observed that UV radiation at 40 mWs/cm2 reduced viral titer by more than 3 log in lettuce and by less than 1.5 log in strawberries. Li et al. (2011) reported that UV light induced a considerable higher reduction (3 log cycle) of murine norovirus-1 (MNV-1) and bacteriophages as a NoV surrogate on stainless steel. In the study of Jean et al. (2011), pulsed

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UV light at 60 mWs/cm2 and 91 mWs/cm2 induced complete reduction (5 log cycle) of MNV-1 and norovirus-1 and HAV, respectively on stainless steel. Pulsed UV light is more effective and rapid infection of inactivating microorganisms than conventional (continuous) UV light (Jean et al., 2011) whereas this radiation technology is expensive (Choudhary and Bandla, 2012). It is well predictable because it depends on the amount of energy delivered and absorbed by the sample. In this respect, pulsed UV light is not different from continuous UV light. There is a need to further examine the disinfection effects of conventional UV-C light against foodborne enteric viruses, including NoV and HAV, specifically on food-contact surfaces because this conventional UV light is environmentally friendly, easy to handle and cost-efficient. Therefore, the present study was to investigate the effectiveness of UV-C radiation (10–300 mWs/cm2) at 260 nm in controlling the infectivity of NoV, using murine norovirus-1 (MNV-1) as an NoV surrogate and an HAV strain on a stainless steel surface, a widely used foodcontact surface that could be a major source of cross-contamination and foodborne enteric viruses and was to compare reduction responses of the viruses on the UV-C treated surface.

onto the surface of the stainless steel coupons using a micropipette. Inoculated coupons were dried in a laminar flow hood for 1 h. Coupon inoculations were performed in triplicate.

2.4. UV-C radiation

2. Materials and methods

UV radiation experiments were performed as described by Ha et al. (2009) to evaluate the effect of UV-C on MNV-1 and HAV on the surface of a stainless steel coupon. A bench-scale, collimated-beam UV reactor equipped with 10-, 15-, and 30-W low-pressure UV lamps (Sankyo Ultraviolet Co., Ltd.; Seoul, Korea) emitting monochromatic UV-C radiation at 260 nm was used in these experiments. UV radiation exposure was measured with an HD 2102.2 photo radiometer (Daehyuntech Co.; Seoul, Korea). The photo radiometer was placed at the same distance (15 cm) as the sample from the UV lamp and calibrated at 260 nm. UV exposure was set at 10, 20, 30, 40, 50, 60, 90, 120, 180, 240, and 300 mWs/cm2. The applied UV dosage (mWs/cm2) was calculated as the exposure time (s) multiplied by the adjusted UV irradiance (μWs/cm2). The UV lamp was turned on at least 30 min prior to the experiments to ensure a constant UV intensity output.

2.1. Virus cell culture

2.5. Sample processing for virus recovery

Murine norovirus-1 (MNV-1; a surrogate for NoV) and Hepatitis A virus (HAV) strain HM-175 were maintained in murine RAW 264.7 cells and monkey FRhK-4 cells, respectively. Cells were cultured in Dulbecco's minimum essential medium (DMEM; Sigma, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Rockville, MD, USA), 44 mM sodium bicarbonate (Sigma, USA), and 1% antibiotic– antimycotic (Penicillin Streptomycin; Gibco, USA), and seeded into 75 cm2 culture flasks for incubation at 37 °C in a humidified incubator containing 5% CO2. The cells were subcultured every two or three days.

Following UV-C radiation, 50 μl of maintenance medium (DMEM + 2% FBS + 44 mM sodium bicarbonate + 1% antibiotic–antimycotic) were deposited on the center of UV-C irradiated stainless steel coupon. The virus-contaminated stainless steel coupon containing the maintenance medium was soaked in 450 μl of the medium in a 15-ml conical tube. The samples were vortexed for 2 min to elute the virus. Each eluted viral suspension was serially diluted 10-fold in DMEM. Stainless steel coupon not expose to UV-C was processed exactly the same way. Log10 reduction values were calculated as follows: log10 reduction = log10 virus titer for the untreated control − log10 virus titer after treatment. Prior to the inactivation study, the amount of viral particles eluted from the surface of stainless steel coupon processing method was determined and considered for calculation of the log10 reduction in inactivation studies. The recovery percentage for each virus was calculated as % recovery = [(Ti / Te) × 100], where Ti = initial viral titer in suspension and Te = vital titer after elution. Viral titers were determined with a plaque assay.

2.2. Virus preparation When the RAW 264.7 and FRhK-4 cell monolayers achieved 90% confluency, the RAW 264.7 cell monolayers were detached using a scraper and FRhK-4 cell monolayers were detached with trypsin. These cells were transferred into a 150-cm2 culture flasks for viral infection. The growth medium was removed by aspiration and the monolayers were washed with phosphate-buffered saline (PBS, pH 7.4). A 200-μl aliquot of the MNV-1 and HAV HM-175 inoculums was added to the flasks, which were incubated for 30 min to allow virus adsorption. The flasks then received 15 ml of maintenance medium (DMEM + 2% FBS + 44 mM sodium bicarbonate + 1% antibiotic–antimycotic), and were incubated at 37 °C in a 5% CO2 atmosphere for 3 days (RAW 264.7) or 7 days (FRhK-4). If the observed cytopathic effects were greater than 90%, the virus-infected flasks were frozen and thawed three times. The viruses were released by cell lysis during this step. The above contents were centrifuged at 1500 ×g for 10 min to remove the cell debris, and the supernatants were subsequently harvested. The viruses were stored at −70 °C until further use. 2.3. Preparation of stainless steel surface material and inoculation Stainless steel (Posco Co., Ltd., SUS 304 2B; Pohang, Korea) was selected as a representative material used in the food industry. Stainless steel coupons measuring Ø 10 mm in diameter and 5 mm in thickness were purchased from a Chung-Ang Scientific Inc. (Seoul, Korea). The coupons were soaked in 70% ethanol for 1 h to remove any residue such as oil and then washed with distilled water. After rinsing, the coupons were dried in a desiccator and placed in a sealed bottle to be autoclaved at 121 °C for 15 min. Fifty microliters of each viral suspension, containing approximately 6.20 log plaque-forming units (PFU)/ml of MNV-1 and 5.85 log PFU/ml of HAV, was inoculated

2.6. Virus titration The MNV-1 and HAV titrations were performed as previously described, with minor modifications as suggested by Wobus et al. (2004) and Bidawid et al. (2000), respectively. Briefly, confluent RAW 264.7 cells or FRhK-4 cells were seeded into each well of a 12-well plate and incubated at 37 °C with 5% CO2 for 36 h (RAW 264.7) or 72 h (FRhK-4) to reach 90% confluence. Viral suspensions eluted from the samples were serially diluted in the maintenance medium (DMEM + 2% FBS + 44 mM sodium bicarbonate + 1% antibiotic–antimycotic). Serially diluted viral suspensions (100 μl) were used to inoculate the cells. After shaking the plates for 10 min (FMS2, FINEPCR, Korea), they were incubated at 37 °C with 5% CO2. One hour later, 2× Type II agarose (Sigma) supplemented with 2 × DMEM was added to the inoculated cells; each well received 1 ml of the mixture. The above plates were left at room temperature for 20 min and incubated at 37 °C in 5% CO2 for 2–3 days to culture MNV-1 and for 7–8 days to culture HAV. The cells were then fixed with 2 ml of 3.7% formaldehyde for 4 h. The formaldehyde was discarded, and the agarose overlays were removed carefully with tap water. The fixed cells were stained with a 0.1% (w/v) crystal violet solution (Invitrogen; Carlsbad, CA., USA) for 20 min to visualize the plaques. Viral titers were calculated as the number of PFU per milliliter.

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2.7. UV-C inactivation mathematical model

Table 2 Effects of UV-C radiation on the reduction of MNV-1 and HAV on a stainless steel surface.

The modified Gompertz equation was used to fit the microbial inactivation curves of sigmoid shape, including the lag phase, exponential inactivation phase, and resistant phase, with three parameters with biological meaning, such as A, kdm and B using GraphPad Prism software version 5.0 for Windows (GraphPad Software; San Diego, CA, USA). The modified Gompertz equation is expressed as following (Zwietring et al., 1990):    −kdm  e ðB−C Þ þ 1 : y ¼ A exp −exp A Where y (log (Nt/N0)) is the logarithm of the ratio of viable virus titers after UV-C treatment to the initial virus titer, A (NmWs/cm2/N0) is the lower asymptote value for UV-C approaching infinity, kdm is the maximum specific value of the inactivation rate when the virus is inactivated exponentially ((mWs/cm2)−1), B is the duration of the lag phase, C is the treatment dose of UV-C (mWs/cm2), and e is exponential. Decimal reduction dose (dR-value) is defined as the UV-C irradiation dose in mWs/cm2 needed to achieve the first decimal reduction of the infectious virus on the inactivation curves at given ranges of UV-C doses and is calculated based on the above equation using Microsoft Excel for Windows. 2.8. Statistical analysis Recovery (%) of MNV-1 and HAV from the stainless steel were analyzed with t-tests using the SAS statistical software program (SAS version 9.1; Cary, NC, USA). Results of anti-viral tests were transformed into log values. One-way ANOVA was used to analyze the virus reduction values among UV-C radiation treatments using the SAS program version 6.11 (SAS Institute Inc., USA). When significant, differences among UV-C radiation treatments were compared using Duncan's multiple range test. In addition, virus reduction values from the same dose of UV-C and dR-values from the two viruses were analyzed with t-tests using the SAS statistical software program. Mean values represent the average of three replicate samples. Statistical significance was set at p b 0.05. 3. Results 3.1. Effects of UV-C radiation on MNV-1 activity on a stainless steel surface Recovery of MNV-1 from the untreated stainless steel surface was 83.5% (Table 1). To assess the effects of UV-C radiation against MNV-1 virus present on a stainless steel surface, the log 10 reductions of MNV-1 with different doses of UV-C were analyzed, as shown in Table 2. The log10 reductions of MNV-1 gradually increased (p b 0.05) with higher doses of UV-C (10–300 mWs/cm2) (Table 2). No significant difference (p N 0.05) in log10 reduction of MNV-1 was observed with 50–60 mWs/cm2 of UV-C on the stainless steel surface. More than 2 log-reductions (N 99%) were observed after 40 mWs/cm2 of UV-C. Unfortunately, 1 log or close to 1-reduction value of MNV-1 was not observed the experimental UV-C radiation doses used in this study.

Table 1 Recovery of MNV-1 and HAV from a stainless steel surface.

MNV-1 HAV

75

Ti (log10 PFU/coupon)

Te (log10 PFU/coupon)

Recovery (%)

6.2 ± 0.1 5.9 ± 0.2

5.2 ± 0.1 4.7 ± 0.1

83.5 ± 0.7 79.5 ± 0.3

Ti: initial viral titer, Te: vital titer after elution, recovery (%) = [(Te / Ti) × 100]. The data presents means of three samples with standard deviations (3 samples/ treatment).

UV-C radiation (mWs/cm2)

Log10 reduction of MNV-1

Log10 reduction of HAV

10 20 30 40 50 60 90 120 180 240 300

0.0 ± 0.0 a, I 0.1 ± 0.0 a, HI 0.3 ± 0.1 a,H 2.0 ± 0.0 a, G 2.3 ± 0.1 a, F 2.5 ± 0.0 a, F 2.9 ± 0.1 a, E 3.7 ± 0.0 a, D 3.9 ± 0.0 a, C 4.2 ± 0.0 a, B 4.4 ± 0.2 a, A

0.0 ± 0.0 a, H 0.1 ± 0.0 a, GH 0.2 ± 0.1 a, G 0.7 ± 0.0 b, F 0.9 ± 0.1 b, E 1.1 ± 0.1 b, D 1.8 ± 0.1 b, C 1.9 ± 0.0 b, C 2.1 ± 0.0 b, B 2.2 ± 0.1 b, B 2.6 ± 0.1 b, A

The data presents means of three samples with standard deviations (3 samples/ treatment). Within the same raw, virus log reduction means with different letters (a or b for each UV-C radiation dose) differ significantly (p b 0.05) by t-test. Within the same column, virus log reduction means with different letters (A ~ I for MNV-1 or A ~ H for HAV) differ significantly (p b 0.05) by Duncan's multiple range test.

Data for MNV-1 survival on the stainless steel surface irradiated by UV-C were fitted using the modified Gompertz equation, which is commonly used for non-linear microbial survival (Fig. 1A); the dR-value of the irradiation dose was predicted using the modified Gompertz equation (Table 3). The modified Gompertz equation fit the experimental data well for all the UV-tested doses (10–300 mWs/cm2), as reflected by an R2 value 0.95 and root mean square error (RMSE) 0.038 (Fig. 1A, Table 3). The dR-value was 33.3 mWs/cm2. 3.2. Effects of UV-C radiation on HAV activity on a stainless steel surface Recovery of HAV from untreated stainless steel surface was 79.4% (Table 1). The log10 reductions of HAV gradually increased (p b 0.05) at higher doses of UV-C (10–300 mWs/cm2) (Table 2). However, there were no significant differences (p N 0.05) in HAV log10 reduction for UV-C radiation at 90–120 or 180–240 mWs/cm2. In addition, 1–2 logreductions of HAV-1 were observed at 60–120 mWs/cm2 of UV-C among the variety of doses of UV-C irradiation. More than 2 logreductions (N 99%) were observed only after 180 mWs/cm2 of UV-C. The HAV survival data from the UV-C-irradiated stainless steel surface were also fitted using the modified Gompertz equation (Fig. 1B); the dR-values of the irradiation dose were predicted using the modified Gompertz equation (Table 3). The modified Gompertz equation fit the experimental data well for all the UV-tested doses (10–300 mWs/ cm2), as evident from an R2 value of 0.97 and RMSE 0.016 (Fig. 1B, Table 3). The dR-value 55.4 for HAV was somewhat similar to the experimental result, with a 1.1 log reduction in the 60 mWs/cm2 UV-Cirradiated stainless steel. These data clearly demonstrate that HAV inactivation by UV-C radiation on the stainless steel was successfully modeled using the modified Gompertz equation. 3.3. Comparison of MNV-1 and HAV recovery, reduction and dR-values after UV-C There was no significant (p N 0.05) difference of recovery from the untreated stainless steel surface between MNV-1 and HAV (Table 1). The log10 reductions of MNV-1 and HAV on the stainless steel surface treated by different doses of UV-C are also shown in Table 2. There was no significant (p N 0.05) difference of log10 reductions between MNV-1 and HAV at 10–30 mWs/cm2 of UV-C. However, the log10 reduction of MNV-1 was significantly (p b 0.05) larger than that of HAV at 40–300 mWs/cm2 of UV-C. Specifically, there were 3.0-, 2.6-, 2.2-, 1.6-, 1.9-, 1.9-, 1.9-, and 1.7-fold increases in the log10 reduction of MNV-1 irradiated with 40, 50, 60, 90, 120, 180, 240, and 300 mWs/cm2 of UV-C, respectively, relative to those of HAV. Moreover, the dR-values of MNV-1 were significantly (p b 0.05) smaller than HAV; there was a 0.6-fold decrease in the dR-values of MNV-1 irradiated with UV-C relative

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Fig. 1. Fitted survival curves for MNV-1 (A) and HAV (B) on a stainless steel surface irradiated by 0–300 mWs/cm2 of UV-C at 260 nm using the modified Gompertz model.

to those of HAV. These data suggest that MNV-1 is generally more sensitive to UV-C than HAV.

4. Discussion Several studies have demonstrated that viruses, including NoV and HAV, persist on environmental surfaces for long periods of time, leading to cross-contamination (Abad et al., 2001; Boone and Gerba, 2007; Mbithi et al., 1991). According to Lamhoujeb et al. (2008), human NoVs can persist on stainless steel from 1 to more than 7 weeks. Moreover, recently our previous studies reported that the infectivity of MNV-1, as a human NoV surrogate (Kim et al., 2014), and HAV (Bae et al., 2014) on all food contact-surfaces (ceramic, wood, rubber, glass, stainless steel, and plastic) persisted after 28 days. After 28 days, MNV-1 and HAV were only reduced by 2.28 and 2.30 log10 PFU/ml, respectively, on the stainless steel surface. UV radiation is a promising technology for surface decontamination in the food processing industry because it is safe, does not leave any residue, does not have legal restrictions, and does not require extensive, expensive equipment for use (Wong et al., 1998; Yousef and Marth, 1988). Many inactivation studies have reported the effectiveness of UV radiation against nonpathogenic spoilage and pathogenic bacteria in diverse foods such as liquid food and meat surfaces. There are some studies of the antiviral effects of continuous UV-C radiation on foods (Fino and Kniel, 2008) and food-related environments (Dee et al., 2011; Jean et al., 2011; Li et al., 2011; Rönnqvist et al., 2014). According to the study of Jean et al. (2011), pulsed light in the wavelength range

Table 3 The dR-values (mWs/cm2 of UV-C) of MNV-1 and HAV on a stainless steel surface exposed to UV-C radiation. Virus

Parameter of Modified Gompertz Equation

MNV-1

B1 ± S.D. kdm2 ± S.D. A3 ± S.D. Correlation coefficient (R2) Root mean square error (RMSE) dR-values of UV-C doses (mWs/cm2) B ± S.D. kdm ± S.D. A ± S.D. Correlation coefficient (R2) Root mean square error (RMSE) dR-values of UV-C doses (mWs/cm2)

HAV

16.520 ± 4.579 −0.060 ± 0.009 −4.029 ± 0.146 0.95 0.038 33.3 ± 7.2b 21.061 ± 3.580 −0.029 ± 0.004 −2.266 ± 0.064 0.97 0.016 55.4 ± 7.5a

dR-value means with different letters (a or b) differ significantly (p b 0.05) by t-test. 1 B is the duration of the lag phase. 2 kdm is the maximum specific value of the inactivation rate when the virus is inactivated exponentially ((mWs/cm2)−1). 3 A (N mWs/cm2/N0) is the lower asymptote value for UV-C approaching infinity.

200–1100 nm for inactivation of enteric viruses could be feasible, especially for the decontamination of stainless steel. However, there is still need more information on the antiviral effects of continuous UV irradiation with diverse food contact surfaces and materials as a substrate. Therefore, this study examined the effects of lower (≤ 300 mWs/cm2) doses of UV-C on MNV-1- and HAV-contaminated stainless steel surfaces as one of the major food contact surfaces. Because there is no cell culture system or animal model for human NoVs (Straub et al., 2007), it is necessary to use a surrogate virus to assess NoV characteristics. Murine norovirus-1 (MNV-1) belongs to the same genus as human NoVs (Brandsma et al., 2012) and can be replicated in cell culture (Wobus et al., 2004). Therefore, it has become a more suitable surrogate virus than feline calicivirus (FCV) (Wobus et al., 2006). Until 2004, FCV was considered the most suitable model for human NoV studies. However, FCV is a respiratory virus and has low tolerance to acidic pH, in contrast with enteric viruses (Doultree et al., 1999). MNV-1 has more similar characteristics to NoVs, including genetic organization (Wobus et al., 2006) and transmission by the fecal–oral route (Wobus et al., 2004). Christopher et al. (2010) reported that 400 mWs/cm2 UV-C at 254 nm induces a more than 5 log-reduction of Salmonella spp., Listeria monocytogenes, and Staphylococcus aureus on stainless steel surfaces. They also reported that 2.52 log-reduction of Salmonella spp. and 2.27 log-reduction of L. monocytogenes were observed at 50 mWs/cm2 UV-C. These reduction values were comparable to the results of the present study, with a 2.34 log-reduction of MNV-1 at 50 mWs/cm2 UV-C and less than 1 log-reduction of HAV at the same UV-C dose. In another study of Dee et al. (2011), porcine reproductive and respiratory syndrome virus (PRRSV) as a pathogen of pigs on the surface of metal, a common material in swine farm was not greatly reduced by UV-C light for 60 min at 254 nm; approx. 0.12-log reduction of PRRSV was revealed by the UV treatment. However, Rönnqvist et al. (2014) recently observed a 4-log reduction of MNV-1 when the virus was exposed to UV-C radiation (60 mWs/cm2) at 253.7 nm on glass slides. Li et al. (2011) also observed approx. 3-log reduction of MNV-1 when the virus was exposed to UV-C light for 5 min at 254 nm although there was no report of exact UV dose used in their study. These reduction values were higher than the results of the present study, with a 2.5 log-reduction of MNV-1 on a stainless steel surface at 60 mWs/cm2 UV-C at 260 nm and only a 0.14 log-reduction of MNV-1 as well as a 0.09 log-reduction of HAV on fresh chicken breast treated with 60 mWs/cm2 UV-C (Park and Ha, 2015). The rough surfaces of fresh meat tend to shield bacteria (Stermer et al., 1987) and viruses (Fino and Kniel, 2008) from radiation. In general, UV radiation is more effective on smooth, even, and clean food and food-contact surfaces and less effective on uneven and rough food and food-contact surfaces, harboring cracks and crevices (Kim et al., 2002). The antimicrobial activity of UV radiation may depend on the type of microorganism, its strain/species, and the surface topography of the food product or food-contact surface. The mechanism behind the antiviral effect of UV-C on stainless steel surface has not been fully

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elucidated. Viral inactivation or reduction by UV radiation is generally considered to be the result of chemical alternations in the RNA or DNA structure, including the formation of pyrimidine dimers (Blactchley and Peel, 2001). However, there is no general rule defining the resistance of viruses to UV radiation based on nucleic acid structure (de Roda Husman et al., 2004). Microbial inactivation is commonly modeled using a first-order kinetics process (Fujikawa and Itoh, 1996; Whiting et al., 1996). More recently, studies suggested that the Weibull model may provide a better fit than first-order models for kinetic analyses of UV light (Bialka et al., 2008; Chun et al., 2010; Park and Ha, 2015). Furthermore, the modified Gompertz equation among non-linear models has been successfully used for modeling the inactivation of L. monocytogenes at isothermal conditions (Linton et al., 1995; Xiong et al., 1999) and Salmonella typhimurium by dense phase carbon dioxide (Liao et al., 2010). In this study, three models were examined to determine the best fit to describe UV-C inactivation of MNV-1 and HAV on a stainless steel surface. The first-order kinetics model was not suitable to demonstrate MNV-1 (Y = − 0.0155X – 0.7521, R2 = 0.77) and HAV (Y = − 0.0091X– 0.03013, R2 = 0.83) inactivation by UV-C treatment, because the R2 values were all much lower than 0.90 (data not shown). Moreover, the Weibull models were not most suitable for modeling the inactivation of MNV-1 and HAV by UV-C data, given that the inactivation curves had both a shoulder and a tail although survival curves of the MNV-1 and HAV fitted with the non-linear Weibull model were concaveupward (n b 1) with the good fit (R2 = 0.91 and RMSE = 0.014 for MNV-1; R2 = 0.95 and RMSE = 0.031 for HAV) (data not shown). However, survival curves of the MNV-1 and HAV were shown as the sigmoid inactivation shape with a shoulder and tail and thus were fitted by the modified Gompertz equation. Therefore, the data fit the modified Gompertz equation (R2 = 0.95 and RMSE = 0.038 for MNV-1; R2 = 0.97 and RMSE = 0.016 for HAV) better than the first-order kinetics model and Weibull model. Thus, to determine d R -values, we only used the modified Gompertz equation as the best fit to the UV-C inactivation data. In the present study, 33.3 mWs/cm 2 for MNV-1 and 55.4 mWs/cm 2 for HAV, as the d R by the modified Gompertz equation were predicted between 30 (0.3 log10 reduction) and 40 mWs/cm2 (2.0 log10 reduction) of UV-C. For the further practical application of the nonlinear regression model of modified Gompertz equation developed for MNV-1 and HAV survival against UV-C light (UV-C dose dependence of the parameters) should be more studied and validated by many experimental samples and diverse UV-C doses; the present study used only 3 samples of stainless steel coupon at each dose of UV-C (10–300 mWs/cm2) for the model fitting and therefore, more data are needed to obtain reliable results for this virus and more studies should be carried out to evaluate the applicability of the non-linear modified Gompertz equation. Nevertheless, the modified Gompertz equation generally could offer a rapid, convenient and reasonable estimate of how an organism will behave in diverse food contact surfaces and foods (for example, estimating UV-C irradiation dose for 5-log reduction value, 5D). Based on the log10 reduction (MNV-1; 0.0–4.4 log-reduction, HAV; 0.0–2.6 log-reduction) and dR-values (MNV-1; 33.3 mWs/cm2, HAV; 55.4 mWs/cm2) of two viruses HAV is more resistant to UV-C radiation (10–300 mWs/cm2) on a stainless steel surface than MNV-1. This is in agreement with data published by Jean et al. (2011), who reported that MNV-1 and HAV on stainless steel were completely inactivated by pulsed light treatment with 0.060 Ws/cm2 and 0.091 Ws/cm2, respectively, and Fraisse et al. (2011), who reported that 100 ppm of peroxyacetic-based biocide on lettuce was found to effectively inactivate MNV-1 (2.3 log-reduction) and HAV (0.7 log-reduction). Fraisse et al. (2011) also noted that HAV is relatively resistant to inactivation by chemical and physical treatments, and thus could provide a good model for determining the efficacy of biocide-based virucidal treatments.

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5. Conclusions The data presented in the present study demonstrate that UV-C radiation has an antiviral effect on the NoV surrogate, MNV-1, and HAV strains on stainless steel surfaces. On these surfaces, UV-C radiation at 10–300 mWs/cm2 reduced MNV-1 and HAV to 0.0–4.4 and 0.0–2.6 log10PFU/ml. The dR-values (first decimal reduction) of MNV-1 (33.3 mWs/cm2) using the modified Gompertz equation were significantly (p b 0.05) lower than those of HAV (55.4 mWs/cm2). These findings show that HAV is more resistant to UV-C radiation, and that the use of low doses of UV-C light on food contact surfaces for inactivation of human NoV and HAV could be feasible in restaurant, institutional, and industrial kitchens and facilities. Acknowledgments This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education(2015R1D1A4A01). 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