Food Environ Virol DOI 10.1007/s12560-014-9154-4
ORIGINAL PAPER
Survival of Norovirus Surrogate on Various Food-Contact Surfaces An-Na Kim • Shin Young Park • San-Cheong Bae Mi-Hwa Oh • Sang-Do Ha
•
Received: 17 February 2014 / Accepted: 20 May 2014 Ó Springer Science+Business Media New York 2014
Abstract Norovirus (NoV) is an environmental threat to humans, which spreads easily from one infected person to another, causing foodborne and waterborne diseases. Therefore, precautions against NoV infection are important in the preparation of food. The aim of this study was to investigate the survival of murine norovirus (MNV), as a NoV surrogate, on six different food-contact surfaces: ceramic, wood, rubber, glass, stainless steel, and plastic. We inoculated 105 PFU of MNV onto the six different surface coupons that were then kept at room temperature for 28 days. On the food-contact surfaces, the greatest reduction in MNV was 2.28 log10 PFU/coupon, observed on stainless steel, while the lowest MNV reduction was 1.29 log10 PFU/coupon, observed on wood. The rank order of MNV reduction, from highest to lowest, was stainless steel, plastic, rubber, glass, ceramic, and wood. The values of dR (time required to reduce the virus by 90 %) on survival plots of MNV determined by a modified Weibull model were 277.60 h (R2 = 0.99) on ceramic, 492.59 h (R2 = 0.98) on wood, 173.56 h on rubber (R2 = 0.98), 97.18 h (R2 = 0.94) on glass, 91.76 h (R2 = 0.97) on stainless steel, and 137.74 h (R2 = 0.97) on plastic. The infectivity of MNV on all food-contact surfaces remained after 28 days. These results show that MNV persists in an infective state on various food-contact surfaces for long
A.-N. Kim S. Y. Park S.-C. Bae S.-D. Ha (&) School of Food Science and Technology, Chung-Ang University, 72–1 Nae-Ri, Daedeok-Myun, Ansung 456-756, Kyunggido, South Korea e-mail: [email protected] M.-H. Oh National Institute of Animal Science, Rural Development Administration, 564 Omockchen-dong, Suwon 441-706, Gyunggido, Republic of Korea
periods. This study may provide valuable information for the control of NoV on various food-contact surfaces, in order to prevent foodborne disease. Keywords Norovirus Survival Food-contact surface Weibull model
Introduction The Sacllan et al. (2011) has reported that norovirus (NoV), hepatitis A virus, rotavirus, sapovirus, and astrovirus are all major pathogens that cause foodborne illnesses in the United States. Virus-related food borne outbreaks may be spread by foods as a result of direct or indirect contamination of the foods with fecal material (Mattison et al. 2007b). Among the enteric viruses, NoV is the leading cause of outbreaks related to food. NoV genus from the Caliciviridae family is the main cause of viral gastroenteritis globally (Siebenga et al. 2009), and it is associated with person-to-person, food, and waterborne outbreaks (Hewitt et al. 2009). The infective dose of NoV is usually very few, sometimes as few as 10 particles (Moe et al. 1999). NoV outbreaks can occur through crosscontamination, especially by food handlers and on the surface of utensils, in contaminated food preparation areas. Fresh produce may be contaminated with the virus due to the use of contaminated irrigation and wash water, infected food handlers, and contact with contaminated surfaces (Anderson et al. 2001; Seymour and Appleton 2001). Other studies have reported that surfaces and fomites contaminated with fecal material, or serving as a repository for aerosolized vomitus, can cause foodborne transmission and subsequent disease outbreaks (Levy et al. 1975; Mbithi et al. 1991; Patterson et al. 1997). In addition, NoV can survive on
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various environmental surfaces and this can lead to cross contamination. NoV is believed to maintain infectivity even after extended periods under different environmental conditions (Rze_zutka and Cook 2004), and is highly persistent on environmental surfaces for a long time (Liu et al. 2012). Boone and Gerba (2007) reported that the persistence of viruses on surfaces depends on many complex variables involving viral properties, such as type and strain, surrounding environment, and surface properties including porosity, state of cleanliness, and the presence of moisture. Although there are some studies regarding the survival of NoV on food-contact surfaces (Giard et al. 2010; Escudero et al. 2012; Liu et al. 2012), it is still needed further information regarding the survival of NoV on diverse types of food-contact surfaces during storage. Because there is no cell culture system or animal model for NoV (Straub et al. 2007), it is necessary to use a surrogate to study virus characteristics. Murine norovirus (MNV) can be replicated in cell culture (Wobus et al. 2004) and therefore has become a more suitable surrogate virus than the previously used feline calicivirus (FCV) (Jean et al. 2011; Steinmann et al. 2008; Wobus et al. 2006). Until 2004, FCV was considered as the most suitable model for NoV studies by 2004. However, FCV is a respiratory virus and has low tolerance to acidic pH, in contrast with enteric viruses (Doultree et al. 1999). MNV has more similar characteristics such as genetic organization (Wobus et al. 2006) and transmission by the fecal-oral route (Wobus et al. 2004). The objective of this study was to investigate the survival of NoV using a NoV surrogate, MNV, on a range of different food-contact surfaces: ceramic, wood, rubber, glass, stainless steel, and plastic, during several days of storage at room temperature.
Materials and Methods Virus and Cell Lines MNV was provided by Dr. Skip Virgin, Washington University. The mouse leukemic monocyte macrophage cell line (RAW 264.7) was purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). Cell Preparation RAW 264.7 cells were grown in Dulbecco’s minimum essential medium (DMEM; SIGMA, USA; cat. # M0268), supplemented with 10 % fetal bovine serum (FBS; Gibco, USA; cat. # 26140-097), 44 mM sodium bicarbonate (SIGMA, USA; cat. # S5761), and 1 % antibiotic–antimycotic (Penicillin Streptomycin; Gibco, USA; cat. #
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15140-122), seeded in 75 cm2 culture flasks, and incubated at 37 °C in a humidified 5 % CO2 incubator. Cells were subcultured every 2 or 3 days. Virus Preparation When monolayers of RAW 264.7 in 150-cm2 culture flasks were 90 % confluent, the growth medium was removed by aspiration. The monolayers were washed with phosphate buffered saline (PBS, pH 7.4). A 1 ml aliquot of virus inoculum was added to each flask, and the flasks were incubated at 37 °C in a 5 % CO2 atmosphere for 90 min to allow virus adsorption. The flasks then received 25 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. If cytopathic effects (CPE) were observed above 90 %, the virus-infected flasks were frozen and thawed three times. Viruses were released by cell lysis through this step. The content was centrifuged at 1,5009g for 10 min to remove cell debris, and the supernatants harvested. And then viruses were stored at -70 °C until used. Plaque Assay RAW 264.7 cells were seeded into each well of 12-well plates and incubated at 37 °C with 5 % CO2 (Seeding volume: 2 ml for each well; 4 9 105 cells). They were incubated for 24 h until the cells reached 90 % confluency. Virus suspensions eluted from the samples were serially diluted in maintenance medium (DMEM ? 2 % FBS ? 44 mM sodium bicarbonate ? 1 % antibiotic–antimycotic). Serially diluted virus suspensions (100 ll) were inoculated onto cells. After shaking the plates for 10 min using a shaker (FMS2, FINEPCR, Korea), they were incubated at 37 °C with 5 % CO2. One hour later, 29 Type II agarose (Sigma) supplemented with 29 DMEM was added to inoculated cells, each well received 1–2 ml of this mixture. These plates were left at room temperature for 20 min and incubated for 2–3 days at 37 °C in 5 % CO2. 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 by tap water. The fixed cells were stained with 0.1 % (w/v) crystal violet solution for 20 min for visualizing the plaques. Plaques were counted, and the virus infectivity titer was described as plaque forming units (PFU) per ml. Preparation of Food-Contact Surface Materials and Inoculation Stainless steel (Posco Co., Ltd., SUS 304 2B, Pohang, Korea), plastic (HDPE; Daesung Industry Co., Seoul,
Food Environ Virol
Korea), wood (Heilongjiang Zhongji IMP & EXP, Beijing, China), rubber (KOMAX Indstrial Co., Ltd., Seoul, Korea), glass (Kukjeglass, Nonsan, Korea), and ceramic (HankookChinaware Co., Ltd., Cheongju, Korea) were selected as representative materials used in the food industry. Coupons with dimensions of Ø 10 mm 9 5 mm were produced with the selected materials. The prepared coupons were soaked in 70 % ethanol as disinfectant for 1 h and washed with distilled water. After rinsing, the coupons were dried in a desiccator and placed in a sealed bottle for autoclaving at 121 °C for 15 min. In the case of rubber, this coupon was not autoclaved because rubber is very heat-sensitive material. The coupon was just soaked in 70 % ethanol for 1 h, washed with distilled water, and dried in a desiccator prior to use in the experiments. Each viral suspension, at 50 ll volume, containing approximately 5 log10 PFU/ml of MNV, was inoculated onto the surface of the materials using a micropipette. Measurement of Viral Survival on Various Materials Inoculated coupons were dried in a laminar flow hood for 1 h and stored for predetermined times (0 min, 3, 12, 24 h, 3, 5, 7, 14, 21, and 28 days) in the experimental chamber at room temperature. Virus recovery rate (%) was calculated with the following formula: % recovery rate = (virus titer from the food-contact surface after 0 h 7 virus titer inoculated on food-contact surface) 9 100 %. Each of coupons was taken out at the point of determined times, and 50 ll of elution buffer (0.05 M glycine–0.14 M NaCl buffer (pH 7.5)) was pipetted onto the coupons and left at room temperature for 10 min. The coupons were placed into 15 ml conical tubes with 200 ll of elution buffer and vortexed for 10 min to elute virus. Then, each eluted viral suspension was 10-fold serially diluted and analyzed by plaque assay. Weibull Model The Weibull model, a two parameters non-linear model, can be expressed as Nt Log ð1Þ ¼ btn N0 Here, Nt is the concentration of virus (PFU/ml) after an exposure time t, N0 is the initial concentration of virus (PFU/ml), t is the exposure time, b and n are the scale (a characteristic time) and the shape parameter as a behavior index, respectively (van Boekel 2002). The b value represents the time needed to reduce the population by one log unit, while the n parameter indicates the shape of the survival curve. An n value of 1 corresponds to a linear survival curve, while n values [1 and \1 correspond to downward and upward concavity, respectively. For the
calculation of dR (analogous to the traditional D value) from the Weibull parameters, Eq. (2) was used, as by Buzrul and Alpas (2007): 1=n 1 dR ¼ ð2Þ b Here, dR = time required to reduce virus by 90 %. To determine inactivation kinetics, the modified Weibull model was fitted by nonlinear regression using the software GraphPad Prism version 5.0 for Windows (GraphPad Software, San Diego, CA, USA). Statistical Analysis For statistical analysis, we used the Statistical Analysis System software version 9.2 (SAS Institute, Cary, NC, USA). Differences among means were examined with Duncan’s multiple range tests, and differences at C5 % level were considered significant.
Results and Discussion Survival of Murine Norovirus on Food-Contact Surfaces The survival of MNV was measured at predetermined times [0 h, 5 h, 12 h, 24 h, 72 h (3 days), 120 h (5 days), 168 h (7 days), 336 h (14 days), 504 h (21 days), and 672 h (28 days)], after inoculation on ceramic, wood, rubber, glass, stainless steel, and plastic. The amount of MNV detected from the surfaces of all six materials significantly decreased over time (Fig. 1). Viral infectivity of MNV on all of the food-contact surfaces was apparent after 28 days. For the surfaces of ceramic, rubber, and glass, there was an initial sharp drop of about 0.4–0.6 log10 PFU/coupon in the MNV titer between 24 and 72 h, and an additional 0.3–0.4 log10 PFU/coupon of inactivation occurred between 120 and 168 h, followed by a steady decline. On plastic coupons, MNV infectivity rapidly decreased after 3 h, followed by relatively stable titers through to the 72 h sampling time point, and a steady reduction after 120 h. The MNV titer from the stainless steel decreased throughout the 28 days, and especially after 72 h, there was a large reduction. In the case of wood, the virus slowly decreased during the whole experimental period. After 672 h (28 days), the greatest reduction in MNV was on stainless steel (2.28 log10 PFU/coupon), while the smallest reduction was seen on wood (1.29 log10 PFU/ coupon). In the cases of ceramic, rubber, glass, and plastic, the values were reduced to 1.58, 2.10, 2.08, and 2.19 log10 PFU/coupon, respectively. From this data, it can be seen
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Fig. 1 Survival of MNV on six food-contact surfaces during the storage time. a Ceramic, b wood, c rubber, d glass, e stainless steel, f plastic
that MNV became inactive much more readily on stainless steel, compared to the other materials, and that it retained its infectivity longer on wood than on the other materials. In this study, SUS 304 2B including copper was used as the
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stainless steel surface. Rifkind et al. (1976) reported that copper ions have the ability to damage DNA by binding to and cross linking between and within strands. If similar mechanisms occurred with the negative-sense RNA
Food Environ Virol Table 1 MNV recovery rates from six food-contact surfaces (ceramic, wood, rubber, glass, stainless steel, plastic) Time (h)
Recovery (%) a–d
Surface types (log10 PFU/coupon) Ceramic
Wood
Rubber
Glass
Stainless steel
Plastic
69.52 ± 0.82b
60.92 ± 0.88d
71.82 ± 0.32a
64.55 ± 1.45c
68.63 ± 0.00b
64.86 ± 0.35c
Means in the same column are significantly (P \ 0.05) different by Duncan’s multiple range test
genome in norovirus, then viral replication could be inhibited by copper-linked RNA damage. There was a statistically significant difference in the viral recovery rates from the six food-contact surfaces at the 0 h point (P \ 0.05), even though an inoculation titer of 5.00 log10 PFU/ml of MNV was used on each surfaces (Table 1). The viral recovery rate from wood appeared to be the lowest value among the materials used, and this can be explained by the fact that the wood absorbed the virus suspension during the inoculation step. Thus, even though the wood coupons seemed to be dried thoroughly after drying for 1 h, the inner region of the wood still remained wet, and retained most of the virus. Additionally, since surface roughness was highest in wood, and its surface is more porous than the other materials (Kim et al. 2011), MNV recovery rates were reduced. Liu et al. (2003) reported an environmental persistence of NoV, as viral RNA has been detected on environmental surfaces including sinks, for several days after initial contamination. FCV remained infectious for 7 and for 20 days at ambient temperature on glass (Doultree et al. 1999) and stainless steel (Mattison et al. 2007a), respectively. D’Souza et al. (2006) demonstrated that NoV can persist on stainless steel surfaces, ceramic, and Formica, for up to 7 days at room temperature at high relative humidity. More recently, Lamhoujeb et al. (2009) suggested that NoV retains its putative infectivity for 56 and 49 days on polyvinyl chloride (PVC) and stainless steel, respectively. Weibull Modeling to Obtain Survival Curves and Parameters Survival data from the food-contact surfaces were fitted using the Weibull model that is used for non-linear microbial survival (Fig. 2). The parameters (b, n, dR, R2) of the Weibull model are shown in Table 2. The goodness of fit of the model was estimated by R2, and since all of the values were over 0.90, this confirmed that the model was a good fit to the survival curves. The first-order kinetic model was also fitted to compare the applicability of the two models (first-order kinetic model and Weibull model). Although the first-order kinetic model is commonly used as a predictive model for microbial reduction by thermal treatment, it was reported to be not suitable for non-thermal treatment and non-linear survival plots (Lee et al. 2009).
Fig. 2 Fits of Weibull models for MNV survival on six food-contact surfaces (ceramic, wood, rubber, glass, stainless steel, plastic)
There have been a number of models proposed to describe non-linear survival curves, and Weibull models have become widely used because of their simplicity and flexibility (Chen and Hoover 2004). A Weibull model assumes that cells and spores in a population retain different resistances, and that a survival curve is just the cumulative form of a distribution of lethal agents (Chen and Hoover 2004). This model is a two parameter model (b, n) and since these parameters are affected by external conditions such as temperature, pH, and the presence of a preservative (Peleg and Cole 2000; Mattick et al. 2001); the Weibull model is used to predict survival curves and calculate the value of dR. The R2 values from the first-order kinetic model and Weibull model were between 0.82–0.96 and 0.94–0.99, respectively. This indicates that the Weibull model fits better than the first-order kinetic model. The dR value calculated from the Weibull parameters (b, n) represents the time required to reduce the virus by 1 log unit. The values obtained were 277.60 h on ceramic, 492.59 h on wood, 173.56 h on rubber, 97.18 h on glass, 91.76 h on stainless steel, and 137.74 h on plastic. From these results, it can be seen that it takes a shorter amount of time to inactivate MNV on the surface of stainless steel than to the other materials. Survival data from this study showed different trends depending on the type of surface material. Variables such as virus type, pH, ionic concentration, surface charge, and organic matter are considered to be responsible for virus attachment to surfaces (Dowd et al. 1998; Grant et al. 1993;
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Food Environ Virol Table 2 Weibull model parameters for MNV survivability on six food-contact surfaces Parameters
Surface types Ceramic
Wood
Rubber
Glass
Stainless steel
Plastic
b ± SE
58.19 ± 11.89
130.20 ± 20.74
36.58 ± 11.19
12.93 ± 8.51
14.81 ± 6.55
26.80 ± 10.75
n ± SE
0.53 ± 0.04
0.63 ± 0.05
0.53 ± 0.05
0.41 ± 0.06
0.45 ± 0.05
0.50 ± 0.06
dR ± SE
277.60 ± 24.31b
492.59 ± 21.62a
173.56 ± 27.76c
97.18 ± 39.04d
91.76 ± 24.35d
137.74 ± 31.06cd
0.99
0.98
0.98
0.94
0.97
0.97
2
R
Values are mean ± standard deviation b = scale parameter, n = shape parameter, concave upward survival curve if n \ 1, concave downward if n [ 1, and linear if n = 1. dR = time (h) required to reduce virus by 90 %, R2 = correlation coefficient, a higher R2 value indicates a better fit to the data a–d
Means in the same column are significantly (P \ 0.05) different by Duncan’s multiple range test
Redman et al. 1997). Electrostatic interactions including van der Waals forces and hydrophobic effects are believed to be involved in the interactions between virus particles and solid substrate (Vega et al. 2005, 2008). The different values from various materials can be explained by the properties of the food-contact surfaces, which affect virus attachment to the surfaces. In addition, if the surfaces were not completely dry, the survival would also be greater (Cannon et al. 2006).
Conclusion In this study, the survival of MNV on different types of food-contact surfaces was investigated. The reduction of MNV was maximal at 2.28 log10 PUF/coupon, on stainless steel, and minimal at 1.29 log10 PUF/coupon, on wood. The rank of reduction of the virus among all materials was in the order of stainless steel, plastic, rubber, glass, ceramic, and wood. The infectivity of the viruses on all food-contact surfaces was retained after 28 days. From our findings, we could know that NoV from food preparation or food handler origin will survive very well on utensils. Thus, it is clear that sanitation in food preparation environments is very important, and that controlled methods for decontaminating NoVcontaminated surfaces are necessary. Reducing the transmission of norovirus from food preparation can therefore be expected to reduce outbreaks related to the virus. Acknowledgments This work was carried out with the support of ‘‘Cooperative Research Program for Agriculture Science & Technology Development (Project No. 009221)’’, Rural Development Administration, Republic of Korea.
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ORIGINAL PAPER
Survival of Norovirus Surrogate on Various Food-Contact Surfaces An-Na Kim • Shin Young Park • San-Cheong Bae Mi-Hwa Oh • Sang-Do Ha
•
Received: 17 February 2014 / Accepted: 20 May 2014 Ó Springer Science+Business Media New York 2014
Abstract Norovirus (NoV) is an environmental threat to humans, which spreads easily from one infected person to another, causing foodborne and waterborne diseases. Therefore, precautions against NoV infection are important in the preparation of food. The aim of this study was to investigate the survival of murine norovirus (MNV), as a NoV surrogate, on six different food-contact surfaces: ceramic, wood, rubber, glass, stainless steel, and plastic. We inoculated 105 PFU of MNV onto the six different surface coupons that were then kept at room temperature for 28 days. On the food-contact surfaces, the greatest reduction in MNV was 2.28 log10 PFU/coupon, observed on stainless steel, while the lowest MNV reduction was 1.29 log10 PFU/coupon, observed on wood. The rank order of MNV reduction, from highest to lowest, was stainless steel, plastic, rubber, glass, ceramic, and wood. The values of dR (time required to reduce the virus by 90 %) on survival plots of MNV determined by a modified Weibull model were 277.60 h (R2 = 0.99) on ceramic, 492.59 h (R2 = 0.98) on wood, 173.56 h on rubber (R2 = 0.98), 97.18 h (R2 = 0.94) on glass, 91.76 h (R2 = 0.97) on stainless steel, and 137.74 h (R2 = 0.97) on plastic. The infectivity of MNV on all food-contact surfaces remained after 28 days. These results show that MNV persists in an infective state on various food-contact surfaces for long
A.-N. Kim S. Y. Park S.-C. Bae S.-D. Ha (&) School of Food Science and Technology, Chung-Ang University, 72–1 Nae-Ri, Daedeok-Myun, Ansung 456-756, Kyunggido, South Korea e-mail: [email protected] M.-H. Oh National Institute of Animal Science, Rural Development Administration, 564 Omockchen-dong, Suwon 441-706, Gyunggido, Republic of Korea
periods. This study may provide valuable information for the control of NoV on various food-contact surfaces, in order to prevent foodborne disease. Keywords Norovirus Survival Food-contact surface Weibull model
Introduction The Sacllan et al. (2011) has reported that norovirus (NoV), hepatitis A virus, rotavirus, sapovirus, and astrovirus are all major pathogens that cause foodborne illnesses in the United States. Virus-related food borne outbreaks may be spread by foods as a result of direct or indirect contamination of the foods with fecal material (Mattison et al. 2007b). Among the enteric viruses, NoV is the leading cause of outbreaks related to food. NoV genus from the Caliciviridae family is the main cause of viral gastroenteritis globally (Siebenga et al. 2009), and it is associated with person-to-person, food, and waterborne outbreaks (Hewitt et al. 2009). The infective dose of NoV is usually very few, sometimes as few as 10 particles (Moe et al. 1999). NoV outbreaks can occur through crosscontamination, especially by food handlers and on the surface of utensils, in contaminated food preparation areas. Fresh produce may be contaminated with the virus due to the use of contaminated irrigation and wash water, infected food handlers, and contact with contaminated surfaces (Anderson et al. 2001; Seymour and Appleton 2001). Other studies have reported that surfaces and fomites contaminated with fecal material, or serving as a repository for aerosolized vomitus, can cause foodborne transmission and subsequent disease outbreaks (Levy et al. 1975; Mbithi et al. 1991; Patterson et al. 1997). In addition, NoV can survive on
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various environmental surfaces and this can lead to cross contamination. NoV is believed to maintain infectivity even after extended periods under different environmental conditions (Rze_zutka and Cook 2004), and is highly persistent on environmental surfaces for a long time (Liu et al. 2012). Boone and Gerba (2007) reported that the persistence of viruses on surfaces depends on many complex variables involving viral properties, such as type and strain, surrounding environment, and surface properties including porosity, state of cleanliness, and the presence of moisture. Although there are some studies regarding the survival of NoV on food-contact surfaces (Giard et al. 2010; Escudero et al. 2012; Liu et al. 2012), it is still needed further information regarding the survival of NoV on diverse types of food-contact surfaces during storage. Because there is no cell culture system or animal model for NoV (Straub et al. 2007), it is necessary to use a surrogate to study virus characteristics. Murine norovirus (MNV) can be replicated in cell culture (Wobus et al. 2004) and therefore has become a more suitable surrogate virus than the previously used feline calicivirus (FCV) (Jean et al. 2011; Steinmann et al. 2008; Wobus et al. 2006). Until 2004, FCV was considered as the most suitable model for NoV studies by 2004. However, FCV is a respiratory virus and has low tolerance to acidic pH, in contrast with enteric viruses (Doultree et al. 1999). MNV has more similar characteristics such as genetic organization (Wobus et al. 2006) and transmission by the fecal-oral route (Wobus et al. 2004). The objective of this study was to investigate the survival of NoV using a NoV surrogate, MNV, on a range of different food-contact surfaces: ceramic, wood, rubber, glass, stainless steel, and plastic, during several days of storage at room temperature.
Materials and Methods Virus and Cell Lines MNV was provided by Dr. Skip Virgin, Washington University. The mouse leukemic monocyte macrophage cell line (RAW 264.7) was purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). Cell Preparation RAW 264.7 cells were grown in Dulbecco’s minimum essential medium (DMEM; SIGMA, USA; cat. # M0268), supplemented with 10 % fetal bovine serum (FBS; Gibco, USA; cat. # 26140-097), 44 mM sodium bicarbonate (SIGMA, USA; cat. # S5761), and 1 % antibiotic–antimycotic (Penicillin Streptomycin; Gibco, USA; cat. #
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15140-122), seeded in 75 cm2 culture flasks, and incubated at 37 °C in a humidified 5 % CO2 incubator. Cells were subcultured every 2 or 3 days. Virus Preparation When monolayers of RAW 264.7 in 150-cm2 culture flasks were 90 % confluent, the growth medium was removed by aspiration. The monolayers were washed with phosphate buffered saline (PBS, pH 7.4). A 1 ml aliquot of virus inoculum was added to each flask, and the flasks were incubated at 37 °C in a 5 % CO2 atmosphere for 90 min to allow virus adsorption. The flasks then received 25 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. If cytopathic effects (CPE) were observed above 90 %, the virus-infected flasks were frozen and thawed three times. Viruses were released by cell lysis through this step. The content was centrifuged at 1,5009g for 10 min to remove cell debris, and the supernatants harvested. And then viruses were stored at -70 °C until used. Plaque Assay RAW 264.7 cells were seeded into each well of 12-well plates and incubated at 37 °C with 5 % CO2 (Seeding volume: 2 ml for each well; 4 9 105 cells). They were incubated for 24 h until the cells reached 90 % confluency. Virus suspensions eluted from the samples were serially diluted in maintenance medium (DMEM ? 2 % FBS ? 44 mM sodium bicarbonate ? 1 % antibiotic–antimycotic). Serially diluted virus suspensions (100 ll) were inoculated onto cells. After shaking the plates for 10 min using a shaker (FMS2, FINEPCR, Korea), they were incubated at 37 °C with 5 % CO2. One hour later, 29 Type II agarose (Sigma) supplemented with 29 DMEM was added to inoculated cells, each well received 1–2 ml of this mixture. These plates were left at room temperature for 20 min and incubated for 2–3 days at 37 °C in 5 % CO2. 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 by tap water. The fixed cells were stained with 0.1 % (w/v) crystal violet solution for 20 min for visualizing the plaques. Plaques were counted, and the virus infectivity titer was described as plaque forming units (PFU) per ml. Preparation of Food-Contact Surface Materials and Inoculation Stainless steel (Posco Co., Ltd., SUS 304 2B, Pohang, Korea), plastic (HDPE; Daesung Industry Co., Seoul,
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Korea), wood (Heilongjiang Zhongji IMP & EXP, Beijing, China), rubber (KOMAX Indstrial Co., Ltd., Seoul, Korea), glass (Kukjeglass, Nonsan, Korea), and ceramic (HankookChinaware Co., Ltd., Cheongju, Korea) were selected as representative materials used in the food industry. Coupons with dimensions of Ø 10 mm 9 5 mm were produced with the selected materials. The prepared coupons were soaked in 70 % ethanol as disinfectant for 1 h and washed with distilled water. After rinsing, the coupons were dried in a desiccator and placed in a sealed bottle for autoclaving at 121 °C for 15 min. In the case of rubber, this coupon was not autoclaved because rubber is very heat-sensitive material. The coupon was just soaked in 70 % ethanol for 1 h, washed with distilled water, and dried in a desiccator prior to use in the experiments. Each viral suspension, at 50 ll volume, containing approximately 5 log10 PFU/ml of MNV, was inoculated onto the surface of the materials using a micropipette. Measurement of Viral Survival on Various Materials Inoculated coupons were dried in a laminar flow hood for 1 h and stored for predetermined times (0 min, 3, 12, 24 h, 3, 5, 7, 14, 21, and 28 days) in the experimental chamber at room temperature. Virus recovery rate (%) was calculated with the following formula: % recovery rate = (virus titer from the food-contact surface after 0 h 7 virus titer inoculated on food-contact surface) 9 100 %. Each of coupons was taken out at the point of determined times, and 50 ll of elution buffer (0.05 M glycine–0.14 M NaCl buffer (pH 7.5)) was pipetted onto the coupons and left at room temperature for 10 min. The coupons were placed into 15 ml conical tubes with 200 ll of elution buffer and vortexed for 10 min to elute virus. Then, each eluted viral suspension was 10-fold serially diluted and analyzed by plaque assay. Weibull Model The Weibull model, a two parameters non-linear model, can be expressed as Nt Log ð1Þ ¼ btn N0 Here, Nt is the concentration of virus (PFU/ml) after an exposure time t, N0 is the initial concentration of virus (PFU/ml), t is the exposure time, b and n are the scale (a characteristic time) and the shape parameter as a behavior index, respectively (van Boekel 2002). The b value represents the time needed to reduce the population by one log unit, while the n parameter indicates the shape of the survival curve. An n value of 1 corresponds to a linear survival curve, while n values [1 and \1 correspond to downward and upward concavity, respectively. For the
calculation of dR (analogous to the traditional D value) from the Weibull parameters, Eq. (2) was used, as by Buzrul and Alpas (2007): 1=n 1 dR ¼ ð2Þ b Here, dR = time required to reduce virus by 90 %. To determine inactivation kinetics, the modified Weibull model was fitted by nonlinear regression using the software GraphPad Prism version 5.0 for Windows (GraphPad Software, San Diego, CA, USA). Statistical Analysis For statistical analysis, we used the Statistical Analysis System software version 9.2 (SAS Institute, Cary, NC, USA). Differences among means were examined with Duncan’s multiple range tests, and differences at C5 % level were considered significant.
Results and Discussion Survival of Murine Norovirus on Food-Contact Surfaces The survival of MNV was measured at predetermined times [0 h, 5 h, 12 h, 24 h, 72 h (3 days), 120 h (5 days), 168 h (7 days), 336 h (14 days), 504 h (21 days), and 672 h (28 days)], after inoculation on ceramic, wood, rubber, glass, stainless steel, and plastic. The amount of MNV detected from the surfaces of all six materials significantly decreased over time (Fig. 1). Viral infectivity of MNV on all of the food-contact surfaces was apparent after 28 days. For the surfaces of ceramic, rubber, and glass, there was an initial sharp drop of about 0.4–0.6 log10 PFU/coupon in the MNV titer between 24 and 72 h, and an additional 0.3–0.4 log10 PFU/coupon of inactivation occurred between 120 and 168 h, followed by a steady decline. On plastic coupons, MNV infectivity rapidly decreased after 3 h, followed by relatively stable titers through to the 72 h sampling time point, and a steady reduction after 120 h. The MNV titer from the stainless steel decreased throughout the 28 days, and especially after 72 h, there was a large reduction. In the case of wood, the virus slowly decreased during the whole experimental period. After 672 h (28 days), the greatest reduction in MNV was on stainless steel (2.28 log10 PFU/coupon), while the smallest reduction was seen on wood (1.29 log10 PFU/ coupon). In the cases of ceramic, rubber, glass, and plastic, the values were reduced to 1.58, 2.10, 2.08, and 2.19 log10 PFU/coupon, respectively. From this data, it can be seen
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Fig. 1 Survival of MNV on six food-contact surfaces during the storage time. a Ceramic, b wood, c rubber, d glass, e stainless steel, f plastic
that MNV became inactive much more readily on stainless steel, compared to the other materials, and that it retained its infectivity longer on wood than on the other materials. In this study, SUS 304 2B including copper was used as the
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stainless steel surface. Rifkind et al. (1976) reported that copper ions have the ability to damage DNA by binding to and cross linking between and within strands. If similar mechanisms occurred with the negative-sense RNA
Food Environ Virol Table 1 MNV recovery rates from six food-contact surfaces (ceramic, wood, rubber, glass, stainless steel, plastic) Time (h)
Recovery (%) a–d
Surface types (log10 PFU/coupon) Ceramic
Wood
Rubber
Glass
Stainless steel
Plastic
69.52 ± 0.82b
60.92 ± 0.88d
71.82 ± 0.32a
64.55 ± 1.45c
68.63 ± 0.00b
64.86 ± 0.35c
Means in the same column are significantly (P \ 0.05) different by Duncan’s multiple range test
genome in norovirus, then viral replication could be inhibited by copper-linked RNA damage. There was a statistically significant difference in the viral recovery rates from the six food-contact surfaces at the 0 h point (P \ 0.05), even though an inoculation titer of 5.00 log10 PFU/ml of MNV was used on each surfaces (Table 1). The viral recovery rate from wood appeared to be the lowest value among the materials used, and this can be explained by the fact that the wood absorbed the virus suspension during the inoculation step. Thus, even though the wood coupons seemed to be dried thoroughly after drying for 1 h, the inner region of the wood still remained wet, and retained most of the virus. Additionally, since surface roughness was highest in wood, and its surface is more porous than the other materials (Kim et al. 2011), MNV recovery rates were reduced. Liu et al. (2003) reported an environmental persistence of NoV, as viral RNA has been detected on environmental surfaces including sinks, for several days after initial contamination. FCV remained infectious for 7 and for 20 days at ambient temperature on glass (Doultree et al. 1999) and stainless steel (Mattison et al. 2007a), respectively. D’Souza et al. (2006) demonstrated that NoV can persist on stainless steel surfaces, ceramic, and Formica, for up to 7 days at room temperature at high relative humidity. More recently, Lamhoujeb et al. (2009) suggested that NoV retains its putative infectivity for 56 and 49 days on polyvinyl chloride (PVC) and stainless steel, respectively. Weibull Modeling to Obtain Survival Curves and Parameters Survival data from the food-contact surfaces were fitted using the Weibull model that is used for non-linear microbial survival (Fig. 2). The parameters (b, n, dR, R2) of the Weibull model are shown in Table 2. The goodness of fit of the model was estimated by R2, and since all of the values were over 0.90, this confirmed that the model was a good fit to the survival curves. The first-order kinetic model was also fitted to compare the applicability of the two models (first-order kinetic model and Weibull model). Although the first-order kinetic model is commonly used as a predictive model for microbial reduction by thermal treatment, it was reported to be not suitable for non-thermal treatment and non-linear survival plots (Lee et al. 2009).
Fig. 2 Fits of Weibull models for MNV survival on six food-contact surfaces (ceramic, wood, rubber, glass, stainless steel, plastic)
There have been a number of models proposed to describe non-linear survival curves, and Weibull models have become widely used because of their simplicity and flexibility (Chen and Hoover 2004). A Weibull model assumes that cells and spores in a population retain different resistances, and that a survival curve is just the cumulative form of a distribution of lethal agents (Chen and Hoover 2004). This model is a two parameter model (b, n) and since these parameters are affected by external conditions such as temperature, pH, and the presence of a preservative (Peleg and Cole 2000; Mattick et al. 2001); the Weibull model is used to predict survival curves and calculate the value of dR. The R2 values from the first-order kinetic model and Weibull model were between 0.82–0.96 and 0.94–0.99, respectively. This indicates that the Weibull model fits better than the first-order kinetic model. The dR value calculated from the Weibull parameters (b, n) represents the time required to reduce the virus by 1 log unit. The values obtained were 277.60 h on ceramic, 492.59 h on wood, 173.56 h on rubber, 97.18 h on glass, 91.76 h on stainless steel, and 137.74 h on plastic. From these results, it can be seen that it takes a shorter amount of time to inactivate MNV on the surface of stainless steel than to the other materials. Survival data from this study showed different trends depending on the type of surface material. Variables such as virus type, pH, ionic concentration, surface charge, and organic matter are considered to be responsible for virus attachment to surfaces (Dowd et al. 1998; Grant et al. 1993;
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Food Environ Virol Table 2 Weibull model parameters for MNV survivability on six food-contact surfaces Parameters
Surface types Ceramic
Wood
Rubber
Glass
Stainless steel
Plastic
b ± SE
58.19 ± 11.89
130.20 ± 20.74
36.58 ± 11.19
12.93 ± 8.51
14.81 ± 6.55
26.80 ± 10.75
n ± SE
0.53 ± 0.04
0.63 ± 0.05
0.53 ± 0.05
0.41 ± 0.06
0.45 ± 0.05
0.50 ± 0.06
dR ± SE
277.60 ± 24.31b
492.59 ± 21.62a
173.56 ± 27.76c
97.18 ± 39.04d
91.76 ± 24.35d
137.74 ± 31.06cd
0.99
0.98
0.98
0.94
0.97
0.97
2
R
Values are mean ± standard deviation b = scale parameter, n = shape parameter, concave upward survival curve if n \ 1, concave downward if n [ 1, and linear if n = 1. dR = time (h) required to reduce virus by 90 %, R2 = correlation coefficient, a higher R2 value indicates a better fit to the data a–d
Means in the same column are significantly (P \ 0.05) different by Duncan’s multiple range test
Redman et al. 1997). Electrostatic interactions including van der Waals forces and hydrophobic effects are believed to be involved in the interactions between virus particles and solid substrate (Vega et al. 2005, 2008). The different values from various materials can be explained by the properties of the food-contact surfaces, which affect virus attachment to the surfaces. In addition, if the surfaces were not completely dry, the survival would also be greater (Cannon et al. 2006).
Conclusion In this study, the survival of MNV on different types of food-contact surfaces was investigated. The reduction of MNV was maximal at 2.28 log10 PUF/coupon, on stainless steel, and minimal at 1.29 log10 PUF/coupon, on wood. The rank of reduction of the virus among all materials was in the order of stainless steel, plastic, rubber, glass, ceramic, and wood. The infectivity of the viruses on all food-contact surfaces was retained after 28 days. From our findings, we could know that NoV from food preparation or food handler origin will survive very well on utensils. Thus, it is clear that sanitation in food preparation environments is very important, and that controlled methods for decontaminating NoVcontaminated surfaces are necessary. Reducing the transmission of norovirus from food preparation can therefore be expected to reduce outbreaks related to the virus. Acknowledgments This work was carried out with the support of ‘‘Cooperative Research Program for Agriculture Science & Technology Development (Project No. 009221)’’, Rural Development Administration, Republic of Korea.
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