- Email: [email protected]
The virucidal effect against murine norovirus and feline calicivirus as surrogates for human norovirus by ethanol-based sanitizers
J Infect Chemother (2013) 19:779–781 DOI 10.1007/s10156-012-0516-2
NOTE
The virucidal effect against murine norovirus and feline calicivirus as surrogates for human norovirus by ethanol-based sanitizers Yuko Shimizu-Onda • Tempei Akasaka • Fumihiro Yagyu Shihoko Komine-Aizawa • Yukinobu Tohya • Satoshi Hayakawa • Hiroshi Ushijima
•
Received: 11 September 2012 / Accepted: 24 October 2012 / Published online: 8 November 2012 Ó Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases 2012
Abstract This study examined the virucidal effects of five types of alcohol-based sanitizers including malic acid and sodium malate, or monoethanolamin, in 58 vol % ethanol (pH 4.0, pH 7.1, pH 11.8), 65 vol % ethanol (pH 4.2), and 75 vol % ethanol (pH 4.4) against murine norovirus (MNV) and feline calicivirus (FCV). The virus titer of MNV was reduced in an ethanol dose-dependent manner under the same pH (about 4.0) condition. Virucidal effect against MNV was correlated with pH when the concentration of ethanol was constant (58 vol %). All the ethanolbased sanitizers provided sufficient virucidal effects against FCV. In conclusion, the virucidal effect of the ethanol-based sanitizer at low concentration of ethanol against norovirus (NoV) is increased when the pH is adjusted to a neutral state. Keywords Murine norovirus Feline calicivirus Surrogate Ethanol-based sanitizer Y. Shimizu-Onda S. Komine-Aizawa S. Hayakawa H. Ushijima (&) Division of Microbiology, Department of Pathology and Microbiology, Nihon University School of Medicine, 30-1 Oyaguchi-Kamicho, Itabashi-ku, Tokyo 173-8610, Japan e-mail: [email protected] T. Akasaka Department of Research and Development, Niitaka Co., Ltd, Osaka, Japan F. Yagyu Department of Developmental Medical Sciences, Institute of International Health, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan Y. Tohya Department of Veterinary Medicine, College of Bioresource Sciences, Nihon University, Kanagawa, Japan
Human norovirus (HuNoV) is a single-strand, positive-sense RNA, and non-enveloped virus that belongs to the family Caliciviridae, genus Norovirus [1]. It causes acute gastroenteritis with the main symptoms of diarrhea, vomiting, and fever [1–3]. There are several routes of infection, including food-borne infection, commonly acquired from eating raw bivalve shellfish. Recently outbreaks have been increased by consuming food that is cooked by infected food handlers. In addition, HuNoV can spread quickly from person to person as well as from contaminated environmental surfaces such as toilets, hands, and cookware [1–3]. Currently, there is no vaccine to prevent the norovirus infection. Therefore, it is important to heat food, to wash or disinfect the contaminated materials, and to practice hand-washing for the prevention of food-borne HuNoV infection. Keeping the hands clean through improved hand hygiene is one of the most important steps to avoid illness and the spreading of HuNoV to others. It is difficult to develop and evaluate antiviral drugs and sanitizers for HuNoV because an efficient culture system for HuNoV has not yet been established [4]. In this study, murine norovirus (MNV) and feline calicivirus (FCV) were used as surrogate viruses for HuNoV. FCV belongs to the same family, Caliciviridae, as HuNoV and is classified into the genus Vesivirus [5]. MNV belongs to genogroup V of the genus Norovirus and is the most similar to HuNoV among viruses that are eligible to be cultured [6–8]. Although sodium hypochlorite and glutaraldehyde are effective disinfectants for HuNoV, they are restricted for use in humans because of their toxicity, whereas ethanol-based sanitizers dry quickly, are harmless for humans, and are suitable for the disinfection of cookware. In this study the virucidal effects of five alcohol-based sanitizer preparations, including malic acid and sodium malate, or monoethanolamin, which have different concentrations of ethanol and different pH, were reported by using MNV and FCV.
123
780
J Infect Chemother (2013) 19:779–781
The 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 three freeze–thaw cycles. The supernatant was centrifuged at 800 g for 15 min, then filtered through a 0.2-lm cellulose acetate membrane and stored at -80 °C. Crandell–Reese feline kidney cells (CRFK: ATCC CCL-94) were inoculated with FCV (F4 strain). After a CPE was observed, cells were treated with three freeze–thaw cycles, and the supernatant was stored at -80 °C after centrifugation at 800 g for 15 min. Five kinds of ethanol-based sanitizers, 58 vol % ethanol (pH 4.0, adjusted by malic acid and sodium malate) (Safecohol 58S), 58 vol % ethanol (pH 7.1, adjusted by malic acid and sodium malate), 58 vol % ethanol (pH 11.8, adjusted by monoethanolamin), 65 vol % ethanol (pH 4.2, adjusted by malic acid and sodium malate), and 75 vol % ethanol (pH 4.4, adjusted by malic acid and sodium malate), were obtained from Niitaka Co. (Osaka, Japan). The stability of pH was examined by the storage stability test. 100 ll virus stock solution was added to 900 ll ethanol-based sanitizers and incubated for 0, 0.5, 1, 2, 5, and 10 min at room temperature. Then, to the 100 ll of MNV and FCV mixtures, 9.9 ml Dulbecco’s modified Eagle’s medium (DMEM) with 10 % fetal bovine serum and 9.9 ml modified Eagle’s medium (MEM) were added, respectively, to stop the reaction. It was previously confirmed that a 100-fold dilution stopped the effects of all kinds of ethanol-based sanitizers but did not affect cells. Then, 50 ll of 10-fold serial dilutions of the samples by DMEM or MEM were inoculated on a 96-well plate in which the RAW 264.7 or CRFK cells were grown to confluence. The plates were incubated at 37 °C in 5 % CO2 for 4 days. The experiment was performed in triplicate. The virus titer was determined as the 50 % tissue culture
infectious dose (TCID50). Phosphate-buffered saline (PBS) was used as the negative control. The virus titer of MNV and FCV was 5.2 log10 TCID50/ 50 ll and 4.7 log10 TCID50/50 ll, respectively. By ethanol-based sanitizer at 75 vol % ethanol (pH 4.4), the titer of MNV was reduced to 3.9 log10 TCID50 at 0.5 min, 4.6 log10 TCID50 at 1 min, and reached the limit of detection beyond 2 min. The virucidal effect of 65 vol % ethanol (pH 4.2) was 3.9 log10 TCID50 at 5 min and 4.5 log10 TCID50 at 10 min. The virus titer was reduced by 58 vol % ethanol (pH 4.0) in a time-dependent manner. However, the reduction was 1.7 log10 TCID50 at 10 min (Fig. 1a). By 58 vol % ethanol (pH 7.1) and 58 vol % ethanol (pH 11.8), the titer was reduced by more than 4 log10 TCID50 at 0.5 min, and reached the limit of detection beyond 1 min (Fig. 2a). On the other hand, the virucidal effects against FCV of all ethanol-based sanitizers reached the limit of detection at 0.5 min at least 4.2 log10 TCID50 (Figs. 1b, 2b). The United States Environmental Protection Agency (EPA) suggests that the test agent is acceptable if there is a minimum of 4-log or 3-log reduction in the CPE of FCV [9]. The virucidal effect against MNV of ethanol-based sanitizers [75 vol % ethanol (pH 4.4), 58 vol % ethanol (pH 11.8), and 58 vol % ethanol (pH 7.1] was more than 3.9 log10 TCID50 at 0.5 min; 65 vol % ethanol (pH 4.2) reduced the virus titer by 3.9 log10 TCID50 at 5 min (Figs. 1a, 2a). All the ethanolbased sanitizers reduced the virus titer by more than 4.0 log10 TCID50 at 0.5 min against FCV (Figs. 1b, 2b). Therefore, according to this standard of the EPA, it is clearly demonstrated that all ethanol-based sanitizers that were tested in this study, except for 58 vol % ethanol (pH 4.0), have a virucidal effect against both MNV and FCV because their reduction of the virus titer was more than 3.9 log10 TCID50 within 5 min. It is reported that the virucidal effect of ethanol
Fig. 1 Virucidal effects of different concentrations and pH of ethanol: 58 vol % (pH 4.0), 65 vol % (pH 4.2), and 75 vol % (pH 4.4). Virus titer of murine norovirus (MNV) (a) and feline calicivirus
(FCV) (b) was determined as the 50 % tissue culture infectious dose (TCID50) at 0, 0.5, 1, 2, 5, and 10 min. PBS, phosphate-buffered saline
123
J Infect Chemother (2013) 19:779–781
781
Fig. 2 Virucidal effects of different pH levels of ethanol: 58 vol % (pH 4.0), 58 vol % (pH 7.1), and 58 vol % (pH 11.8). Virus titer of MNV (a) and FCV (b) was determined as the 50 % tissue culture infectious dose (TCID50) at 0, 0.5, 1, 2, 5, and 10 min
against MNV was dependent on the concentrations [10–13]. In this study, under the same pH (weak acid) condition, the virus titer of MNV was reduced in an ethanol dose-dependent manner (Fig. 1a). Under the condition of the same concentration of ethanol (58 %), the virucidal effects of the ethanolbased sanitizer 58 vol % ethanol (pH 4.0) was 1.8 log10 TCID50 at 10 min; on the other hand, the virucidal effects of the ethanol-based sanitizers 58 vol % ethanol (pH 11.8) and 58 vol % ethanol (pH 7.1) were more than 4.0 log10 TCID50 within 0.5 min (Fig. 2a). Consequently, the virucidal effect against MNV was correlated with the pH. This is the first report that shows virucidal effects of ethanol-based sanitizers at an alkali or neutral state against MNV and FCV. Therefore, it is important to rinse detergents and dry cookware to maintain the concentration of ethanol and pH to make ethanol-based sanitizers efficient for kitchen use. In conclusion, the virucidal effect of the ethanol-based sanitizer at low concentration of ethanol against NoV is increased when the pH is adjusted to neutral state. Acknowledgments This research was supported by a Grant-in-Aid from the Ministry of Education, Culture, Sport, Sciences and Technology and the Ministry of Health, Labor and Welfare, Japan. We thank Drs. N. Maeekarn, S. Okitsu, and P. Khamrin for their manuscript advice. Conflict of interest
None.
References 1. Glass RI, Parashar UD, Estes MK. Norovirus gastroenteritis. N Engl J Med. 2009;361(18):1776–85.
2. Center for Disease Control and Prevention. Updated norovirus outbreak management and disease prevention guidelines. MMWR Recomm Rep. 2011;60(3):1–15. 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(10):802–10. 4. Duizer E, Schwab KJ, Neill FH, Atmar RL, Koopmans MPG, Estes MK. Laboratory efforts to cultivate noroviruses. J Gen Virol. 2004;85(pt 1):79–87. 5. Green KY, Belliot G, Taylor JL, et al (2007) Norovirus. In: Fields virology, 5th edn. Lippincott, Williams & Wilkins, Philadelphia, pp 952–953. 6. Wobus CE, Thackray LB, Virgin HW 4th. Murine norovirus: a model system to study norovirus biology and pathogenesis. J Virol. 2006;80(11):5104–12. 7. Karst SM, Wobus CE, Lay M, Davidson J, Virgin HW 4th. STAT1-dependent innate immunity to a Norwalk-like virus. Science. 2003;299(5612):1575–8. 8. 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(12):2076–84. 9. U.S. Environmental Protection Agency: Confirmatory virucidal effectiveness test using feline calicivirus as surrogate for norovirus. http://www.epa.gov/oppad001/pdf_files/ confirmatory_virucidal_test.pdf. 10. 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(6):388–94. 11. 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(12):2232–8. 12. 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(10):3315–8. 13. Paulmann D, Steinmann J, Becker B, Steinmann E, Steinmann J. Virucidal activity of different alcohols against murine norovirus, a surrogate of human norovirus. J Hosp Infect. 2011;79:378–82.
123
NOTE
The virucidal effect against murine norovirus and feline calicivirus as surrogates for human norovirus by ethanol-based sanitizers Yuko Shimizu-Onda • Tempei Akasaka • Fumihiro Yagyu Shihoko Komine-Aizawa • Yukinobu Tohya • Satoshi Hayakawa • Hiroshi Ushijima
•
Received: 11 September 2012 / Accepted: 24 October 2012 / Published online: 8 November 2012 Ó Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases 2012
Abstract This study examined the virucidal effects of five types of alcohol-based sanitizers including malic acid and sodium malate, or monoethanolamin, in 58 vol % ethanol (pH 4.0, pH 7.1, pH 11.8), 65 vol % ethanol (pH 4.2), and 75 vol % ethanol (pH 4.4) against murine norovirus (MNV) and feline calicivirus (FCV). The virus titer of MNV was reduced in an ethanol dose-dependent manner under the same pH (about 4.0) condition. Virucidal effect against MNV was correlated with pH when the concentration of ethanol was constant (58 vol %). All the ethanolbased sanitizers provided sufficient virucidal effects against FCV. In conclusion, the virucidal effect of the ethanol-based sanitizer at low concentration of ethanol against norovirus (NoV) is increased when the pH is adjusted to a neutral state. Keywords Murine norovirus Feline calicivirus Surrogate Ethanol-based sanitizer Y. Shimizu-Onda S. Komine-Aizawa S. Hayakawa H. Ushijima (&) Division of Microbiology, Department of Pathology and Microbiology, Nihon University School of Medicine, 30-1 Oyaguchi-Kamicho, Itabashi-ku, Tokyo 173-8610, Japan e-mail: [email protected] T. Akasaka Department of Research and Development, Niitaka Co., Ltd, Osaka, Japan F. Yagyu Department of Developmental Medical Sciences, Institute of International Health, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan Y. Tohya Department of Veterinary Medicine, College of Bioresource Sciences, Nihon University, Kanagawa, Japan
Human norovirus (HuNoV) is a single-strand, positive-sense RNA, and non-enveloped virus that belongs to the family Caliciviridae, genus Norovirus [1]. It causes acute gastroenteritis with the main symptoms of diarrhea, vomiting, and fever [1–3]. There are several routes of infection, including food-borne infection, commonly acquired from eating raw bivalve shellfish. Recently outbreaks have been increased by consuming food that is cooked by infected food handlers. In addition, HuNoV can spread quickly from person to person as well as from contaminated environmental surfaces such as toilets, hands, and cookware [1–3]. Currently, there is no vaccine to prevent the norovirus infection. Therefore, it is important to heat food, to wash or disinfect the contaminated materials, and to practice hand-washing for the prevention of food-borne HuNoV infection. Keeping the hands clean through improved hand hygiene is one of the most important steps to avoid illness and the spreading of HuNoV to others. It is difficult to develop and evaluate antiviral drugs and sanitizers for HuNoV because an efficient culture system for HuNoV has not yet been established [4]. In this study, murine norovirus (MNV) and feline calicivirus (FCV) were used as surrogate viruses for HuNoV. FCV belongs to the same family, Caliciviridae, as HuNoV and is classified into the genus Vesivirus [5]. MNV belongs to genogroup V of the genus Norovirus and is the most similar to HuNoV among viruses that are eligible to be cultured [6–8]. Although sodium hypochlorite and glutaraldehyde are effective disinfectants for HuNoV, they are restricted for use in humans because of their toxicity, whereas ethanol-based sanitizers dry quickly, are harmless for humans, and are suitable for the disinfection of cookware. In this study the virucidal effects of five alcohol-based sanitizer preparations, including malic acid and sodium malate, or monoethanolamin, which have different concentrations of ethanol and different pH, were reported by using MNV and FCV.
123
780
J Infect Chemother (2013) 19:779–781
The 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 three freeze–thaw cycles. The supernatant was centrifuged at 800 g for 15 min, then filtered through a 0.2-lm cellulose acetate membrane and stored at -80 °C. Crandell–Reese feline kidney cells (CRFK: ATCC CCL-94) were inoculated with FCV (F4 strain). After a CPE was observed, cells were treated with three freeze–thaw cycles, and the supernatant was stored at -80 °C after centrifugation at 800 g for 15 min. Five kinds of ethanol-based sanitizers, 58 vol % ethanol (pH 4.0, adjusted by malic acid and sodium malate) (Safecohol 58S), 58 vol % ethanol (pH 7.1, adjusted by malic acid and sodium malate), 58 vol % ethanol (pH 11.8, adjusted by monoethanolamin), 65 vol % ethanol (pH 4.2, adjusted by malic acid and sodium malate), and 75 vol % ethanol (pH 4.4, adjusted by malic acid and sodium malate), were obtained from Niitaka Co. (Osaka, Japan). The stability of pH was examined by the storage stability test. 100 ll virus stock solution was added to 900 ll ethanol-based sanitizers and incubated for 0, 0.5, 1, 2, 5, and 10 min at room temperature. Then, to the 100 ll of MNV and FCV mixtures, 9.9 ml Dulbecco’s modified Eagle’s medium (DMEM) with 10 % fetal bovine serum and 9.9 ml modified Eagle’s medium (MEM) were added, respectively, to stop the reaction. It was previously confirmed that a 100-fold dilution stopped the effects of all kinds of ethanol-based sanitizers but did not affect cells. Then, 50 ll of 10-fold serial dilutions of the samples by DMEM or MEM were inoculated on a 96-well plate in which the RAW 264.7 or CRFK cells were grown to confluence. The plates were incubated at 37 °C in 5 % CO2 for 4 days. The experiment was performed in triplicate. The virus titer was determined as the 50 % tissue culture
infectious dose (TCID50). Phosphate-buffered saline (PBS) was used as the negative control. The virus titer of MNV and FCV was 5.2 log10 TCID50/ 50 ll and 4.7 log10 TCID50/50 ll, respectively. By ethanol-based sanitizer at 75 vol % ethanol (pH 4.4), the titer of MNV was reduced to 3.9 log10 TCID50 at 0.5 min, 4.6 log10 TCID50 at 1 min, and reached the limit of detection beyond 2 min. The virucidal effect of 65 vol % ethanol (pH 4.2) was 3.9 log10 TCID50 at 5 min and 4.5 log10 TCID50 at 10 min. The virus titer was reduced by 58 vol % ethanol (pH 4.0) in a time-dependent manner. However, the reduction was 1.7 log10 TCID50 at 10 min (Fig. 1a). By 58 vol % ethanol (pH 7.1) and 58 vol % ethanol (pH 11.8), the titer was reduced by more than 4 log10 TCID50 at 0.5 min, and reached the limit of detection beyond 1 min (Fig. 2a). On the other hand, the virucidal effects against FCV of all ethanol-based sanitizers reached the limit of detection at 0.5 min at least 4.2 log10 TCID50 (Figs. 1b, 2b). The United States Environmental Protection Agency (EPA) suggests that the test agent is acceptable if there is a minimum of 4-log or 3-log reduction in the CPE of FCV [9]. The virucidal effect against MNV of ethanol-based sanitizers [75 vol % ethanol (pH 4.4), 58 vol % ethanol (pH 11.8), and 58 vol % ethanol (pH 7.1] was more than 3.9 log10 TCID50 at 0.5 min; 65 vol % ethanol (pH 4.2) reduced the virus titer by 3.9 log10 TCID50 at 5 min (Figs. 1a, 2a). All the ethanolbased sanitizers reduced the virus titer by more than 4.0 log10 TCID50 at 0.5 min against FCV (Figs. 1b, 2b). Therefore, according to this standard of the EPA, it is clearly demonstrated that all ethanol-based sanitizers that were tested in this study, except for 58 vol % ethanol (pH 4.0), have a virucidal effect against both MNV and FCV because their reduction of the virus titer was more than 3.9 log10 TCID50 within 5 min. It is reported that the virucidal effect of ethanol
Fig. 1 Virucidal effects of different concentrations and pH of ethanol: 58 vol % (pH 4.0), 65 vol % (pH 4.2), and 75 vol % (pH 4.4). Virus titer of murine norovirus (MNV) (a) and feline calicivirus
(FCV) (b) was determined as the 50 % tissue culture infectious dose (TCID50) at 0, 0.5, 1, 2, 5, and 10 min. PBS, phosphate-buffered saline
123
J Infect Chemother (2013) 19:779–781
781
Fig. 2 Virucidal effects of different pH levels of ethanol: 58 vol % (pH 4.0), 58 vol % (pH 7.1), and 58 vol % (pH 11.8). Virus titer of MNV (a) and FCV (b) was determined as the 50 % tissue culture infectious dose (TCID50) at 0, 0.5, 1, 2, 5, and 10 min
against MNV was dependent on the concentrations [10–13]. In this study, under the same pH (weak acid) condition, the virus titer of MNV was reduced in an ethanol dose-dependent manner (Fig. 1a). Under the condition of the same concentration of ethanol (58 %), the virucidal effects of the ethanolbased sanitizer 58 vol % ethanol (pH 4.0) was 1.8 log10 TCID50 at 10 min; on the other hand, the virucidal effects of the ethanol-based sanitizers 58 vol % ethanol (pH 11.8) and 58 vol % ethanol (pH 7.1) were more than 4.0 log10 TCID50 within 0.5 min (Fig. 2a). Consequently, the virucidal effect against MNV was correlated with the pH. This is the first report that shows virucidal effects of ethanol-based sanitizers at an alkali or neutral state against MNV and FCV. Therefore, it is important to rinse detergents and dry cookware to maintain the concentration of ethanol and pH to make ethanol-based sanitizers efficient for kitchen use. In conclusion, the virucidal effect of the ethanol-based sanitizer at low concentration of ethanol against NoV is increased when the pH is adjusted to neutral state. Acknowledgments This research was supported by a Grant-in-Aid from the Ministry of Education, Culture, Sport, Sciences and Technology and the Ministry of Health, Labor and Welfare, Japan. We thank Drs. N. Maeekarn, S. Okitsu, and P. Khamrin for their manuscript advice. Conflict of interest
None.
References 1. Glass RI, Parashar UD, Estes MK. Norovirus gastroenteritis. N Engl J Med. 2009;361(18):1776–85.
2. Center for Disease Control and Prevention. Updated norovirus outbreak management and disease prevention guidelines. MMWR Recomm Rep. 2011;60(3):1–15. 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(10):802–10. 4. Duizer E, Schwab KJ, Neill FH, Atmar RL, Koopmans MPG, Estes MK. Laboratory efforts to cultivate noroviruses. J Gen Virol. 2004;85(pt 1):79–87. 5. Green KY, Belliot G, Taylor JL, et al (2007) Norovirus. In: Fields virology, 5th edn. Lippincott, Williams & Wilkins, Philadelphia, pp 952–953. 6. Wobus CE, Thackray LB, Virgin HW 4th. Murine norovirus: a model system to study norovirus biology and pathogenesis. J Virol. 2006;80(11):5104–12. 7. Karst SM, Wobus CE, Lay M, Davidson J, Virgin HW 4th. STAT1-dependent innate immunity to a Norwalk-like virus. Science. 2003;299(5612):1575–8. 8. 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(12):2076–84. 9. U.S. Environmental Protection Agency: Confirmatory virucidal effectiveness test using feline calicivirus as surrogate for norovirus. http://www.epa.gov/oppad001/pdf_files/ confirmatory_virucidal_test.pdf. 10. 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(6):388–94. 11. 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(12):2232–8. 12. 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(10):3315–8. 13. Paulmann D, Steinmann J, Becker B, Steinmann E, Steinmann J. Virucidal activity of different alcohols against murine norovirus, a surrogate of human norovirus. J Hosp Infect. 2011;79:378–82.
123