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The virucidal effects against murine norovirus and feline calicivirus F4 as surrogates for human norovirus by the different additive concentrations of ethanol-based sanitizers
J Infect Chemother 22 (2016) 191e193
Contents lists available at ScienceDirect
Journal of Infection and Chemotherapy journal homepage: http://www.elsevier.com/locate/jic
Note
The virucidal effects against murine norovirus and feline calicivirus F4 as surrogates for human norovirus by the different additive concentrations of ethanol-based sanitizers Tempei Akasaka a, Yuko Shimizu-Onda b, Satoshi Hayakawa b, Hiroshi Ushijima b, * a b
Department of Research and Development, Niitaka Co., Ltd, Osaka, Japan Division of Microbiology, Department of Pathology and Microbiology, Nihon University School of Medicine, Tokyo, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 February 2015 Received in revised form 23 August 2015 Accepted 13 September 2015 Available online 21 October 2015
Since human norovirus is non-cultivable, murine norovirus and feline calicivirus have been used as surrogates. In this study, the virucidal effects of ethanol-based sanitizers with different concentrations of additives (malic acid/sodium malate, glycerin-fatty acid ester) against murine norovirus and feline calicivirus F4 were examined. The ethanol-based sanitizers at pH 7 showed sufficient virucidal effects, but glycerin-fatty acid ester included in ethanol-based sanitizers at pH 4 or 6 reduced the virucidal effects against murine norovirus. The ethanol-based sanitizers containing malic acid/sodium malate inactivated feline calicivirus F4 in shorter time, but there is no difference between ethanol-based sanitizers with and without glycerin-fatty acid ester. Traditionally, feline calicivirus has been used for long time as a surrogate virus for human norovirus. However, this study suggested that murine norovirus and feline calicivirus F4 had different sensitivity with the additive components of ethanol-based sanitizers. Therefore, using feline calicivirus alone as a surrogate for human norovirus may not be sufficient to evaluate the virucidal effect of sanitizers on food-borne infections caused by human norovirus. Sanitizers having virucidal effects against at least both murine norovirus and feline calicivirus may be more suitable to inactivate human norovirus. © 2015, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Keywords: Murine norovirus Feline calicivirus Human norovirus Surrogate Ethanol-based sanitizer Virucidal effect
Human norovirus (HuNoV) is a non-enveloped virus that belongs to the family Caliciviridae, genus Norovirus. It causes acute gastroenteritis with the main symptoms of diarrhea, vomiting and fever [1e3]. HuNoV is the leading cause of food-borne outbreaks and gastroenteritis as reported by the Ministry of Health, Labor and Welfare in Japan in 2013 [4]. Eating raw bivalve shellfish is considered as the main route of HuNoV infection. Outbreaks due to consuming food prepared by infected food handlers have been increased recently and become a significant social problem. HuNoV is very contagious and infection is easily spread by exposure to contaminated environmental surfaces such as toilets, hands, and cookware [1e3]. Therefore, it is important to heat food, to wash and disinfect the contaminated materials, and to practice hand-
* Corresponding author. Division of Microbiology, Department of Pathology and Microbiology, Nihon University School of Medicine, 30-1 Oyaguchi-Kamicho, Itabashi-ku, Tokyo 173-8610, Japan. Tel.: þ81 03 3972 8111; fax: þ81 03 3972 9560. E-mail address: [email protected] (H. Ushijima).
washing in order to prevent food-borne illness and infection caused by HuNoV. It is difficult to develop and evaluate antiviral drugs and sanitizers against HuNoV since an efficient cell culture system for HuNoV has not yet established [5]. Therefore, feline calicivirus (FCV) has been used as a surrogate virus for HuNoV. However, FCV (genus Vesivirus) does not belong to the same genus of HuNoV [6]. Recently murine norovirus (MNV) is noted as a new surrogate for HuNoV among eligibly cultivable viruses since it belongs to the same genus and is most similar to HuNoV [7e9]. Ethanol-based sanitizers dry quickly, and are harmless for humans. Therefore, they are suitable for the disinfection of hand and cookware. We have studied the virucidal effects of the ethanol-based sanitizers against FCV and MNV as surrogates for HuNoV, and we reported previously that the same ethanol-based sanitizers had different virucidal effect on MNV and FCV-F4 [10]. In this report, the influence of different components of ethanol-based sanitizers on virucidal effect was studied. Six types of 50.18% (w/w) ethanol-based sanitizers with malic acid/sodium malate and glycerin-fatty acid
http://dx.doi.org/10.1016/j.jiac.2015.09.002 1341-321X/© 2015, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
192
T. Akasaka et al. / J Infect Chemother 22 (2016) 191e193
Table 1 Samples of ethanol-based sanitizers.
Sample Sample Sample Sample Sample Sample
1 2 3 4 5 6
Ethanol
pH
Malic acid/sodium malate
Glycerin-fatty acid ester
50.18% 50.18% 50.18% 50.18% 50.18% 50.18%
4.1 4.0 7.1 7.2 5.8 6.3
0.35%/0.06% 0.35%/0.06% 0%/0.53% 0%/0.53% e e
0.3% e 0.3% e 0.3% e
ester having different pH were used to evaluate the virucidal effect against MNV and FCV-F4. A murine macrophage-like cells (RAW 264.7:ATCC TIB-71) were inoculated with MNV (S7-PP3 strain). After a cytopathic effect (CPE) was observed, cells were treated with 3 freezeethaw cycles, and centrifuged at 800 g for 15 min. The supernatant was filtered through 0.2 mm cellulose acetate membrane, and stored at 80 C. The titer of MNV was 107.58.0 Tissue Culture Infectious Dose 50% (TCID50)/50 ml. Crandell-Reese feline kidney cells (CRFK:ATCC CCL-94) was inoculated with FCV F4 strain, which was isolated from cats in Japan with respiratory symptoms. After a CPE was observed, cells were treated with 3 freezeethaw cycles, and the supernatant was centrifuged at 800 g for 15 min, then stored at e 80 C. The titer of FCV-F4 was 107.07.5 TCID50/50 ml. Six kinds of 50.18% (w/w) ethanol-based sanitizers having the pH of 4, 6, and 7 adjusted by malic acid, sodium malate, and glycerin-fatty acid ester were used (Table 1). Phosphate-buffered saline (PBS) was used as the negative control. 100 ml virus stock solution was added to 900 ml ethanol-based sanitizers and PBS. The mixtures were incubated for 0, 0.5, 1, 2, 5, and 10 min at room temperature. Then, to stop the reaction, 990 ml of Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum and 990 ml of Modified Eagle Medium (MEM) were added to 10 ml of MNV and FCV-F4 mixtures, respectively. It was previously confirmed that 100 times dilution stopped the effect of all kinds of ethanol-based sanitizers, but did not affect cells. 50 ml of 10-fold serial dilutions of the samples by DMEM or MEM was inoculated on 96-well plate in which the RAW 264.7 or CRFK cells were grown to confluence. The experiment was performed in triplicate and repeated 4 times.
The reduction of the virus titer of MNV was more than 4.0 log10 TCID50/50 ml at 0.5 min by ethanol-based sanitizers with and without glycerin-fatty acid ester, adjusted at pH 7 by malic acid and sodium malate (sample 3 and 4). However, the reduction was 1.8 log10 TCID50/50 ml and 2.5 log10 TCID50/50 ml at 10 min by sample 1 and 2, ethanol-based sanitizers adjusted at pH 4 in the same manner. The reduction by sample 5 and 6 was 3.0 log10 TCID50/50 ml and 3.5 log10 TCID50/50 ml at 10 min. These samples had the pH of 6, having no malic acid and sodium malate (Fig. 1). Therefore, it is clear that glycerin-fatty acid in additives of ethanol-based sanitizers tends to reduce the virucidal effect against MNV. The reduction of the virus titer of FCV-F4 was about 4.0 log10 TCID50/50 ml at 1 min by all test samples. However, sample 5 and 6 without malic acid/sodium malate were less effective against FCVF4 than other samples. They did not reach the limit to detection at 10 min. There was no influence of glycerin-fatty acid ester on the virucidal effect against FCV-F4 (Fig. 2). It was reported that the virucidal effect of ethanol against MNV depended on the concentration of ethanol [11e13]. High content of ethanol may help to inactivate non-enveloped viruses, but it also increases the risk of cytotoxicity and lowers the flash point. Recently, some low concentrated ethanol-based sanitizers with additives are commercially available. The bactericidal effect of these sanitizers was proved. In this study, we examined the virucidal effect of the ethanol-based sanitizer with low ethanol concentration (50.18w/w%). Moreover, our previous study showed that the virucidal effect depended on not only the concentration of ethanol but also the pH of ethanol-based sanitizer [10]. Malic acid and its salts are considered as a good pH adjuster. In addition, they are stable, cheap, and enhance the bactericidal activity. They also have a good safety profile that is suitable to include them in sanitizers. It is well known that glycerin-fatty acid ester in ethanol sanitizer enhances the bactericidal effect [14], but it reduced the virucidal effect against MNV in this study. Glycerin-fatty acid ester is known to affect the cell membrane as an emulsifier and detergent. Therefore, it may be not effective against MNV which has no cell membrane. The reason why glycerin-fatty acid ester reduces the virucidal effect against MNV is still unclear. It is reported that the ethanol-based sanitizers containing organic acid and phosphoric acid enhance the virucidal effect against MS2 phage and FCV [15]. Our study also showed a similar
Fig. 1. The virucidal effects of ethanol-based sanitizers against murine norovirus. One hundred ml of virus stock solution was added into 900 ml of ethanol-based sanitizers, and incubated for 0, 0.5, 1, 2, 5 and 10 min. The virus titer was determined as the 50% tissue culture infectious dose (TCID50)/50 ml.
T. Akasaka et al. / J Infect Chemother 22 (2016) 191e193
193
Fig. 2. The virucidal effects of ethanol-based sanitizers against feline calicivirus F4. One hundred ml of virus stock solution was added into 900 ml of ethanol-based sanitizers, and incubated for 0, 0.5, 1, 2, 5 and 10 min. The virus titer was determined as the 50% tissue culture infectious dose (TCID50)/50 ml.
tendency. Different to MNV, glycerin-fatty acid ester did not change the virucidal effect against FCV-F4. The reasons for the different effects of glycerin-fatty acid ester against FCV-F4 and MNV are not known. Further studies are required to elucidate this phenomenon. In conclusion, our study showed that the components of ethanol-based sanitizer in addition to the pH influenced the virucidal effect. Particularly, glycerin-fatty acid ester inhibited the virucidal effect against MNV, but not against FCV-F4, which was very sensitive to ethanol. Therefore, it is necessary to use not only FCV-F4 but also MNV and different types of FCVs as surrogates for HuNoV, to evaluate the effect of sanitizers against HuNoV. Conflict of interest None. Acknowledgment This study was supported by the Japan Society for the Promotion of Science (JSPS) Grants-in-Aid for Scientific Research (Grant Number 24390266). We would like to thank Drs. Dinh Nguyen Tran, Sayaka Takanashi, Shoko Okitsu and Yukinobu Tohya for their helps in this study. References [1] Glass RI, Parashar UD, Estes MK. Norovirus gastroenteritis. N Engl J Med 2009;361:1776e85. [2] Centers for Disease Control and Prevention. Updated norovirus outbreak management and disease prevention guidelines. MMWR Recomm Rep 2011;60:1e15.
[3] Wu HM, Fornek M, Schwab KJ, Chapin AR, Gibson K, Schwab E, et al. A norovirus outbreak at a long-term-care facility: the role of environmental surface contamination. Infect Cont Hosp Epidemiol 2005;26:802e10. [4] The Ministry of Health, Labor and Welfare. The food-borne illness statistics 2013 in Japan. Available at: http://www.mhlw.go.jp/stf/seisakunitsuite/ bunya/kenkou_iryou/shokuhin/syokuchu/04.html. [accessed 5.11.14]. [5] Duizer E, Schwab KJ, Neill FH, Atmar RL, Koopmans MPG, Estes MK. Laboratory efforts to cultivate noroviruses. J Gen Virol 2004;85:79e87. [6] Green KY. The noroviruses. In: Knipe DM, Howley PM, editors. Fields virology. 5th ed. Philadelphia, PA: Lippincott Williams & Wilkins; 2007. p. 949e79. [7] Wobus CE, Thackray LB, Virgin 4th HW. Murine norovirus: a model system to study norovirus biology and pathogenesis. J Virol 2006;80:5104e12. [8] Karst SM, Wobus CE, Lay M, Davidson J, Virgin 4th HW. STAT1-dependent innate immunity to a Norwalk-like virus. Science 2003;299:1575e8. [9] Wobus CE, Karst SM, Thackray LB, Chang KO, Sosnovtsev SV, Belliot G, et al. Replication of Norovirus in cell culture reveals a tropism for dendritic cells and macrophages. PLoS Biol 2004;2:e432. [10] Shimizu-Onda Y, Akasaka T, Yagyu F, Aizawa S, Tohya Y, Hayakawa S, et al. The virucidal effect against murine norovirus and feline calicivirus as surrogates for human norovirus by ethanol-based sanitizers. J Infect Chemother 2013;19:779e81. [11] Shimizu Y, Ushijima H, Kitajima M, Katayama H, Tohya Y. Murine norovirus as a novel surrogate to evaluate disinfectants for human norovirus. Jpn J Environ Infect 2009;24:388e94 [in Japanese]. [12] Park GW, Barclay L, Macinga D, Charbonneau D, Pettigrew CA, Vinje J. Comparative efficacy of seven hand sanitizers against murine norovirus, feline calicivirus, and GII.4 norovirus. J Food Prot 2010;73:2232e8. [13] Belliot G, Lavaux A, Souihel D, Agnello D, Pothier P. Use of murine norovirus as a surrogate to evaluate resistance of human norovirus to disinfectants. Appl Environ Microbiol 2008;74:3315e8. [14] Fukao T. About the use of alcohol to food. Chemical preservation technique for food. No. 13. Bokin Bobai 2009;37:913e22 [in Japanese]. [15] Matsumura R, Nakamura E, Noguchi E, Kumashita Y, Yamamoto M, Furuta T. Efficacy of virucidal alcohol-based hand antiseptics. Bokin Bobai 2013;41: 421e5 [in Japanese].
Contents lists available at ScienceDirect
Journal of Infection and Chemotherapy journal homepage: http://www.elsevier.com/locate/jic
Note
The virucidal effects against murine norovirus and feline calicivirus F4 as surrogates for human norovirus by the different additive concentrations of ethanol-based sanitizers Tempei Akasaka a, Yuko Shimizu-Onda b, Satoshi Hayakawa b, Hiroshi Ushijima b, * a b
Department of Research and Development, Niitaka Co., Ltd, Osaka, Japan Division of Microbiology, Department of Pathology and Microbiology, Nihon University School of Medicine, Tokyo, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 February 2015 Received in revised form 23 August 2015 Accepted 13 September 2015 Available online 21 October 2015
Since human norovirus is non-cultivable, murine norovirus and feline calicivirus have been used as surrogates. In this study, the virucidal effects of ethanol-based sanitizers with different concentrations of additives (malic acid/sodium malate, glycerin-fatty acid ester) against murine norovirus and feline calicivirus F4 were examined. The ethanol-based sanitizers at pH 7 showed sufficient virucidal effects, but glycerin-fatty acid ester included in ethanol-based sanitizers at pH 4 or 6 reduced the virucidal effects against murine norovirus. The ethanol-based sanitizers containing malic acid/sodium malate inactivated feline calicivirus F4 in shorter time, but there is no difference between ethanol-based sanitizers with and without glycerin-fatty acid ester. Traditionally, feline calicivirus has been used for long time as a surrogate virus for human norovirus. However, this study suggested that murine norovirus and feline calicivirus F4 had different sensitivity with the additive components of ethanol-based sanitizers. Therefore, using feline calicivirus alone as a surrogate for human norovirus may not be sufficient to evaluate the virucidal effect of sanitizers on food-borne infections caused by human norovirus. Sanitizers having virucidal effects against at least both murine norovirus and feline calicivirus may be more suitable to inactivate human norovirus. © 2015, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Keywords: Murine norovirus Feline calicivirus Human norovirus Surrogate Ethanol-based sanitizer Virucidal effect
Human norovirus (HuNoV) is a non-enveloped virus that belongs to the family Caliciviridae, genus Norovirus. It causes acute gastroenteritis with the main symptoms of diarrhea, vomiting and fever [1e3]. HuNoV is the leading cause of food-borne outbreaks and gastroenteritis as reported by the Ministry of Health, Labor and Welfare in Japan in 2013 [4]. Eating raw bivalve shellfish is considered as the main route of HuNoV infection. Outbreaks due to consuming food prepared by infected food handlers have been increased recently and become a significant social problem. HuNoV is very contagious and infection is easily spread by exposure to contaminated environmental surfaces such as toilets, hands, and cookware [1e3]. Therefore, it is important to heat food, to wash and disinfect the contaminated materials, and to practice hand-
* Corresponding author. Division of Microbiology, Department of Pathology and Microbiology, Nihon University School of Medicine, 30-1 Oyaguchi-Kamicho, Itabashi-ku, Tokyo 173-8610, Japan. Tel.: þ81 03 3972 8111; fax: þ81 03 3972 9560. E-mail address: [email protected] (H. Ushijima).
washing in order to prevent food-borne illness and infection caused by HuNoV. It is difficult to develop and evaluate antiviral drugs and sanitizers against HuNoV since an efficient cell culture system for HuNoV has not yet established [5]. Therefore, feline calicivirus (FCV) has been used as a surrogate virus for HuNoV. However, FCV (genus Vesivirus) does not belong to the same genus of HuNoV [6]. Recently murine norovirus (MNV) is noted as a new surrogate for HuNoV among eligibly cultivable viruses since it belongs to the same genus and is most similar to HuNoV [7e9]. Ethanol-based sanitizers dry quickly, and are harmless for humans. Therefore, they are suitable for the disinfection of hand and cookware. We have studied the virucidal effects of the ethanol-based sanitizers against FCV and MNV as surrogates for HuNoV, and we reported previously that the same ethanol-based sanitizers had different virucidal effect on MNV and FCV-F4 [10]. In this report, the influence of different components of ethanol-based sanitizers on virucidal effect was studied. Six types of 50.18% (w/w) ethanol-based sanitizers with malic acid/sodium malate and glycerin-fatty acid
http://dx.doi.org/10.1016/j.jiac.2015.09.002 1341-321X/© 2015, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
192
T. Akasaka et al. / J Infect Chemother 22 (2016) 191e193
Table 1 Samples of ethanol-based sanitizers.
Sample Sample Sample Sample Sample Sample
1 2 3 4 5 6
Ethanol
pH
Malic acid/sodium malate
Glycerin-fatty acid ester
50.18% 50.18% 50.18% 50.18% 50.18% 50.18%
4.1 4.0 7.1 7.2 5.8 6.3
0.35%/0.06% 0.35%/0.06% 0%/0.53% 0%/0.53% e e
0.3% e 0.3% e 0.3% e
ester having different pH were used to evaluate the virucidal effect against MNV and FCV-F4. A murine macrophage-like cells (RAW 264.7:ATCC TIB-71) were inoculated with MNV (S7-PP3 strain). After a cytopathic effect (CPE) was observed, cells were treated with 3 freezeethaw cycles, and centrifuged at 800 g for 15 min. The supernatant was filtered through 0.2 mm cellulose acetate membrane, and stored at 80 C. The titer of MNV was 107.58.0 Tissue Culture Infectious Dose 50% (TCID50)/50 ml. Crandell-Reese feline kidney cells (CRFK:ATCC CCL-94) was inoculated with FCV F4 strain, which was isolated from cats in Japan with respiratory symptoms. After a CPE was observed, cells were treated with 3 freezeethaw cycles, and the supernatant was centrifuged at 800 g for 15 min, then stored at e 80 C. The titer of FCV-F4 was 107.07.5 TCID50/50 ml. Six kinds of 50.18% (w/w) ethanol-based sanitizers having the pH of 4, 6, and 7 adjusted by malic acid, sodium malate, and glycerin-fatty acid ester were used (Table 1). Phosphate-buffered saline (PBS) was used as the negative control. 100 ml virus stock solution was added to 900 ml ethanol-based sanitizers and PBS. The mixtures were incubated for 0, 0.5, 1, 2, 5, and 10 min at room temperature. Then, to stop the reaction, 990 ml of Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum and 990 ml of Modified Eagle Medium (MEM) were added to 10 ml of MNV and FCV-F4 mixtures, respectively. It was previously confirmed that 100 times dilution stopped the effect of all kinds of ethanol-based sanitizers, but did not affect cells. 50 ml of 10-fold serial dilutions of the samples by DMEM or MEM was inoculated on 96-well plate in which the RAW 264.7 or CRFK cells were grown to confluence. The experiment was performed in triplicate and repeated 4 times.
The reduction of the virus titer of MNV was more than 4.0 log10 TCID50/50 ml at 0.5 min by ethanol-based sanitizers with and without glycerin-fatty acid ester, adjusted at pH 7 by malic acid and sodium malate (sample 3 and 4). However, the reduction was 1.8 log10 TCID50/50 ml and 2.5 log10 TCID50/50 ml at 10 min by sample 1 and 2, ethanol-based sanitizers adjusted at pH 4 in the same manner. The reduction by sample 5 and 6 was 3.0 log10 TCID50/50 ml and 3.5 log10 TCID50/50 ml at 10 min. These samples had the pH of 6, having no malic acid and sodium malate (Fig. 1). Therefore, it is clear that glycerin-fatty acid in additives of ethanol-based sanitizers tends to reduce the virucidal effect against MNV. The reduction of the virus titer of FCV-F4 was about 4.0 log10 TCID50/50 ml at 1 min by all test samples. However, sample 5 and 6 without malic acid/sodium malate were less effective against FCVF4 than other samples. They did not reach the limit to detection at 10 min. There was no influence of glycerin-fatty acid ester on the virucidal effect against FCV-F4 (Fig. 2). It was reported that the virucidal effect of ethanol against MNV depended on the concentration of ethanol [11e13]. High content of ethanol may help to inactivate non-enveloped viruses, but it also increases the risk of cytotoxicity and lowers the flash point. Recently, some low concentrated ethanol-based sanitizers with additives are commercially available. The bactericidal effect of these sanitizers was proved. In this study, we examined the virucidal effect of the ethanol-based sanitizer with low ethanol concentration (50.18w/w%). Moreover, our previous study showed that the virucidal effect depended on not only the concentration of ethanol but also the pH of ethanol-based sanitizer [10]. Malic acid and its salts are considered as a good pH adjuster. In addition, they are stable, cheap, and enhance the bactericidal activity. They also have a good safety profile that is suitable to include them in sanitizers. It is well known that glycerin-fatty acid ester in ethanol sanitizer enhances the bactericidal effect [14], but it reduced the virucidal effect against MNV in this study. Glycerin-fatty acid ester is known to affect the cell membrane as an emulsifier and detergent. Therefore, it may be not effective against MNV which has no cell membrane. The reason why glycerin-fatty acid ester reduces the virucidal effect against MNV is still unclear. It is reported that the ethanol-based sanitizers containing organic acid and phosphoric acid enhance the virucidal effect against MS2 phage and FCV [15]. Our study also showed a similar
Fig. 1. The virucidal effects of ethanol-based sanitizers against murine norovirus. One hundred ml of virus stock solution was added into 900 ml of ethanol-based sanitizers, and incubated for 0, 0.5, 1, 2, 5 and 10 min. The virus titer was determined as the 50% tissue culture infectious dose (TCID50)/50 ml.
T. Akasaka et al. / J Infect Chemother 22 (2016) 191e193
193
Fig. 2. The virucidal effects of ethanol-based sanitizers against feline calicivirus F4. One hundred ml of virus stock solution was added into 900 ml of ethanol-based sanitizers, and incubated for 0, 0.5, 1, 2, 5 and 10 min. The virus titer was determined as the 50% tissue culture infectious dose (TCID50)/50 ml.
tendency. Different to MNV, glycerin-fatty acid ester did not change the virucidal effect against FCV-F4. The reasons for the different effects of glycerin-fatty acid ester against FCV-F4 and MNV are not known. Further studies are required to elucidate this phenomenon. In conclusion, our study showed that the components of ethanol-based sanitizer in addition to the pH influenced the virucidal effect. Particularly, glycerin-fatty acid ester inhibited the virucidal effect against MNV, but not against FCV-F4, which was very sensitive to ethanol. Therefore, it is necessary to use not only FCV-F4 but also MNV and different types of FCVs as surrogates for HuNoV, to evaluate the effect of sanitizers against HuNoV. Conflict of interest None. Acknowledgment This study was supported by the Japan Society for the Promotion of Science (JSPS) Grants-in-Aid for Scientific Research (Grant Number 24390266). We would like to thank Drs. Dinh Nguyen Tran, Sayaka Takanashi, Shoko Okitsu and Yukinobu Tohya for their helps in this study. References [1] Glass RI, Parashar UD, Estes MK. Norovirus gastroenteritis. N Engl J Med 2009;361:1776e85. [2] Centers for Disease Control and Prevention. Updated norovirus outbreak management and disease prevention guidelines. MMWR Recomm Rep 2011;60:1e15.
[3] Wu HM, Fornek M, Schwab KJ, Chapin AR, Gibson K, Schwab E, et al. A norovirus outbreak at a long-term-care facility: the role of environmental surface contamination. Infect Cont Hosp Epidemiol 2005;26:802e10. [4] The Ministry of Health, Labor and Welfare. The food-borne illness statistics 2013 in Japan. Available at: http://www.mhlw.go.jp/stf/seisakunitsuite/ bunya/kenkou_iryou/shokuhin/syokuchu/04.html. [accessed 5.11.14]. [5] Duizer E, Schwab KJ, Neill FH, Atmar RL, Koopmans MPG, Estes MK. Laboratory efforts to cultivate noroviruses. J Gen Virol 2004;85:79e87. [6] Green KY. The noroviruses. In: Knipe DM, Howley PM, editors. Fields virology. 5th ed. Philadelphia, PA: Lippincott Williams & Wilkins; 2007. p. 949e79. [7] Wobus CE, Thackray LB, Virgin 4th HW. Murine norovirus: a model system to study norovirus biology and pathogenesis. J Virol 2006;80:5104e12. [8] Karst SM, Wobus CE, Lay M, Davidson J, Virgin 4th HW. STAT1-dependent innate immunity to a Norwalk-like virus. Science 2003;299:1575e8. [9] Wobus CE, Karst SM, Thackray LB, Chang KO, Sosnovtsev SV, Belliot G, et al. Replication of Norovirus in cell culture reveals a tropism for dendritic cells and macrophages. PLoS Biol 2004;2:e432. [10] Shimizu-Onda Y, Akasaka T, Yagyu F, Aizawa S, Tohya Y, Hayakawa S, et al. The virucidal effect against murine norovirus and feline calicivirus as surrogates for human norovirus by ethanol-based sanitizers. J Infect Chemother 2013;19:779e81. [11] Shimizu Y, Ushijima H, Kitajima M, Katayama H, Tohya Y. Murine norovirus as a novel surrogate to evaluate disinfectants for human norovirus. Jpn J Environ Infect 2009;24:388e94 [in Japanese]. [12] Park GW, Barclay L, Macinga D, Charbonneau D, Pettigrew CA, Vinje J. Comparative efficacy of seven hand sanitizers against murine norovirus, feline calicivirus, and GII.4 norovirus. J Food Prot 2010;73:2232e8. [13] Belliot G, Lavaux A, Souihel D, Agnello D, Pothier P. Use of murine norovirus as a surrogate to evaluate resistance of human norovirus to disinfectants. Appl Environ Microbiol 2008;74:3315e8. [14] Fukao T. About the use of alcohol to food. Chemical preservation technique for food. No. 13. Bokin Bobai 2009;37:913e22 [in Japanese]. [15] Matsumura R, Nakamura E, Noguchi E, Kumashita Y, Yamamoto M, Furuta T. Efficacy of virucidal alcohol-based hand antiseptics. Bokin Bobai 2013;41: 421e5 [in Japanese].