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Virucidal Efficacy of Olanexidine Gluconate as a Hand Antiseptic Against Human Norovirus

Virucidal Efficacy of Olanexidine Gluconate as a Hand Antiseptic Against Human Norovirus Human noroviruses are the major cause of non-bacterial acute gastroenteritis worldwide. Since no therapeutic agent has been proven to prevent human norovirus infection yet, preventive healthcare interventions to block the infection routes play an important role in infection control. One of the possible infection routes of human noroviruses are through contaminated hands, but no hand antiseptics have been proven effective. Olanexidine gluconate is a new biguanide compound that has already been approved for sale as an antiseptic for the surgical field in Japan. A new hand antiseptic was developed using olanexidine gluconate in this study, and its virucidal efficacy against human noroviruses was evaluated using modified RT- qPCR that can account for genome derived from intact viruses using RNase A and photo-reactive intercalators. We tested the virucidal efficacy of five materials; two olanexidine gluconate antiseptics (hand rub formulation and surgical field formula- tion), two kinds of ethanol solutions at different pH (approx. 3 or 7), and a base component of olanexidine gluconate hand rub formulation against 11 human norovirus genotypes by culture-independent methods. The infectivity of murine norovirus (MNV), a surrogate for human norovirus, was significantly reduced after use of the antiseptics. The olanexidine gluconate hand rub demonstrated the strongest virucidal efficacy against human norovirus among the five tested materials. This study showed that olanexidine gluconate has the potential to become a strong tool for the prevention of human norovirus infection. Keywords Human norovirus · Inactivation · Olanexidine · Antiseptic · RT-qPCR Introduction (Adams et al. 2016). Of the ten genogroups of norovirus, genogroups I (GI), GII, GIV, GVIII, and GIX can infect Noroviruses are a non-enveloped and single-stranded posi- humans, and GIII, GV, GVII, and GX can infect bovine, tive-sense RNA virus belonging to the family Caliciviridae, mice, dogs, and bats, respectively, (Chhabra et al. 2019). and its particle is an icosahedron with a diameter of 38 nm Infectivity of human norovirus is considered to be very strong; fewer than ten viral particles have been reported adequate to establish infection (Feng et al. 2011). Human Electronic supplementary material The online version of this noroviruses cause acute gastroenteritis in medical facilities, article (https ://doi.org/10.1007/s1256 0-020-09422 -4) contains schools and restaurants, and the economic burden of the ill- supplementary material, which is available to authorized users. ness is $4.2 billion and $60.3 billion in direct medical and indirect societal costs per year, respectively (Bartsch et al. * Daisuke Sano daisuke.sano.e1@tohoku.ac.jp 2016). Varied infection vehicles of human norovirus have been reported, including shellfish (Campos and Lees 2014), Naruto Research Institute, Research and Development fruits (Berger et al. 2010) and other foods such as dried laver Center, Otsuka Pharmaceutical Factory, Inc., Naruto, seaweed (Kusumi et al. 2017). Human noroviruses grow in Tokushima 772-8601, Japan small intestinal epithelial cells after a latency period of 12 Department of Frontier Science for Advanced Environment, to 48 h and cause symptoms such as abdominal pain, diar- Graduate School of Environmental Studies, Tohoku University, Aoba 6-6-06, Aramaki, Aoba-ku, Sendai, rhea, nausea and vomiting (Dolin et al. 1972). Usually the Miyagi 980-8579, Japan symptoms disappear within 2 days, but they may be severe Department of Civil and Environmental Engineering, depending on the physical strength and hygienic condition of Graduate School of Engineering, Tohoku University, Aoba patients (Fischer Walker et al. 2012; Liu et al. 2012). Human 6-6-06, Aramaki, Aoba-ku, Sendai, Miyagi 980-8579, Japan Vol:.(1234567890) 1 3 Food and Environmental Virology (2020) 12:180–190 181 norovirus particles released from the feces of infected people improved version of OLG, ethanol was added to provide are resistant to drying and heat, and maintain infectivity in quick drying, and the pH was adjusted to be alkaline to natural environments, which poses a risk of secondary infec- enhance the capsid denaturation effect. The virucidal effi- tion (Barclay et al. 2015; Lopman et al. 2012; Robilotti et al. cacy of five antiseptics including OLG-HR against eleven 2015). Since no therapeutic agent or vaccine has been proven different genotypes of human norovirus was evaluated by a effective against human noroviruses, hygienic interventions, culture-independent method (RT-qPCR) using RNase A and including hand washing, are regarded important for control photo-reactive intercalator PMAxx (propidium monoazide) of the infection (Barclay et al. 2015; Park et al. 2014; Park pretreatment step. The virucidal effects of OLG-HR were and Sobsey 2011) (Table 1). compared with those of ethanol for disinfection (EtOH), Olanexidine gluconate is a novel antimicrobial compound acidically adjusted ethanol (pH 3) for disinfection (EtOH- developed by Otsuka Pharmaceutical Factory, Inc. and has A), base ingredient excluding olanexidine gluconate from been used in Japan clinically for preoperative skin prepa- OLG-HR (Base), and OLG (Table 2). EtOH was used as a ration [Product name: Olanedine Antiseptic Solution 1.5% test antiseptic because hand sanitizers containing ethanol has (OLG)] (Hagi et al. 2015; Inoue et al. 2015; Nakata et al. been recommended for the disinfection of hands by the Cent- 2017). The virucidal efficacy of OLG against feline calici- ers for Disease Control and Prevention (CDC) and World virus at 60-s and 10 min were 0.60, and 2.85, respectively Health Organization (WHO) (Boyce and Pittet 2002; WHO (unpublished data). Because European Committee for Stand- 2009). EtOH-A was used because of its high inactivation ardization states that “the product shall demonstrate at least efficiency against surrogate viruses, although its efficacy for a decimal log reduction of 4 in virus titre” (EN 14476 2013), human noroviruses were unknown (Park et al. 2010). The the virucidal activity of OLG against a non-enveloped virus disinfection efficiency of OLG-HR and other four antisep - is not enough as well as other biguanide compounds (Iwa- tics against multiple genotypes within GI, GII and GIV of sawa et al. 2012; Sickbert-Bennett et al. 2005). The mode of noroviruses were compared. action of OLG is similar to that of chlorhexidine gluconate, a biguanide antiseptic, but OLG has also been confirmed to have a stronger protein denaturation effect than chlorhex- Results idine gluconate (Hagi et al. 2015). The objective of this study is to develop a new olanex- Virucidal Efficacy of Test Materials Against Murine idine-containing formulation for hand hygiene (olanexidine Norovirus by Modified RT‑qPCR and Plaque Assay gluconate hand rub; OLG-HR) in anticipation of the pro- tein denaturation activity of OLG for destroying the cap- Since we have no simple in vitro culture method for human sid protein of human noroviruses. In OLG-HR, which is an noroviruses, it is difficult to evaluate the correlation between the value of RT-qPCR and the virus infectivity. Therefore, we compared the results from the modified RT-qPCR and Table 1 Genotype and viral titer of norovirus positive stool samples the plaque assay using murine norovirus (MNV) (Table 3). in this study The virucidal efficiency of each test material was similar Genogroup Genotype Quantity (Log except for OLG. The log reduction values (LRVs, ± SD) RNA copies/well) of OLG at 30- and 60-s contact time evaluated using the Human norovirus GI GI.2 4.45 GI.3 5.75 Table 2 Details of antiseptics evaluated in this study GI.4 4.85 Test material Active ingredient pH GI.6 4.07 Name Concentration of GI.7 5.75 olanexidine gluco- Human norovirus GII GII.2 4.52 nate (%) GII.4 Den Haag 4.82 2006b OLG-HR Olanexidine gluco- 1.5 9.5 GII.10 4.17 nate/ethanol (70%) GII.12 4.56 EtOH Ethanol (76.9– – Approx. 7 81.4%) GII.14 4.29 EtOH-A Ethanol (76.9– – Approx. 3 Human norovirus GIV GIV.1 6.16 81.4%) This value was RNA copies number of 100-fold diluted solution of Base Ethanol (70%) – 9.5 test virus solution. This measurement was carried out prior to the OLG Olanexidine gluco- 1.5 5 virucidal activity test in order to confirm the concentration of the nate stool samples 1 3 182 Food and Environmental Virology (2020) 12:180–190 Table 3 Virucidal efficacy of Test material Reaction Log reduction of modified RT-qPCR Log reduction by plaque assay test materials against murine time (s) [log copies/well, mean ± SD, n = 3] [log PFU/mL, mean ± SD, 10 10 norovirus S99 by modified n = 3] RT-qPCR and plaque assay OLG-HR 30 3.92 ± 0.21 > 4.58 60 4.04 ± 0.18 > 4.58 EtOH 30 4.00 ± 0.25 > 4.58 60 3.96 ± 0.17 > 4.58 EtOH-A 30 3.90 ± 0.16 > 4.58 60 4.05 ± 0.22 > 4.58 Base 30 4.05 ± 0.21 > 4.58 60 3.96 ± 0.20 > 4.58 OLG 30 1.90 ± 0.14 2.35 ± 0.22 60 1.83 ± 0.42 2.73 ± 0.80 plaque assay were 2.35 (± 0.22) and 2.73 (± 0.80), and those with OLG-HR were 30- and 60-s, respectively, which using the modified RT-qPCR assay were 1.90 (± 0.14) and were similar to the results of Base. The mean LRVs of 1.83 (± 0.42), respectively. Log reduction obtainable by the EtOH against norovirus GI.4, GI.6, and GI.7 were > 2.34 other four test materials containing ethanol were between log (higher than the quantification limit), > 2.33 log , 10 10 3.90 and 4.04 at both contact times when modified RT-qPCR and 3.20 (± 0.36) log at 30-s, whereas those for noro- assay was used, and those evaluated by the plaque assay virus GI.2 and GI.3 were 0.92 (± 0.31) and 0.10 (± 0.03) were > 4.58. log even after 60-s. EtOH-A was not effective against norovirus GI.3, and the mean LRVs against the 4 other Virucidal Efficacy of Test Materials Against Norovirus GI genotypes were between 1.11 and 1.81 l og at 60-s GI Genotypes (Table 4). The median LRVs of norovirus GI genotypes at 30-s contact time using OLG-HR, EtOH, EtOH-A, Base, The virucidal efficacy of test materials against norovirus and OLG were 2.34, 2.08, 0.74, 2.34, and 1.52 (Fig. 1a), GI genotypes was evaluated using the modified RT-qPCR and the 60-s contact time medians were 2.34, 2.33, 1.25, assay. The RNA copy numbers (log RNA copies/well) of 2.34, and 1.50, respectively (Fig. 1b). The median LRV norovirus GI.2, GI.3, GI.4, GI.6, and GI.7 in PBS (nega- of norovirus GI genotypes was significantly higher when tive control) were between 3.33 to 4.64, and the protein OLG-HR and Base were used at both contact times. The concentrations of five GI stool samples were between 2.19 difference between OLG-HR and Base was not significant and 14.61 mg/mL (Table S1). When OLG-HR was used, at both contact times (Fig. 1). There was no correlation the mean LRVs of norovirus GI.4, GI.6, and GI.7 were between protein concentration of norovirus stool sam- higher than the quantification limit at the 30-s contact ples and LRVs for each test material both 30- and 60-s time (Table 4). The mean LRVs of norovirus GI.2 were (Table S1 and Fig. S1). 1.76 (± 0.18) and 1.70 (± 0.20) when the contact times Table 4 Virucidal efficacy of Test material Reaction Log reduction [mean ± SD, n = 3] test materials against norovirus time (s) GI genotypes GI.2 GI.3 GI.4 GI.6 GI.7 OLG-HR 30 1.76 ± 0.18 3.28 ± 0.54 > 2.34 > 2.33 > 3.34 60 1.70 ± 0.20 > 3.64 > 2.34 > 2.33 > 3.34 EtOH 30 0.72 ± 0.16 0.01 ± 0.25 > 2.34 > 2.33 3.20 ± 0.36 60 0.92 ± 0.31 0.10 ± 0.03 > 2.34 > 2.33 > 3.34 EtOH-A 30 0.99 ± 0.10 0.59 ± 0.07 0.38 ± 0.21 1.24 ± 0.17 0.80 ± 0.25 60 1.16 ± 0.12 0.66 ± 0.02 1.11 ± 0.03 1.63 ± 0.16 1.81 ± 0.33 Base 30 1.75 ± 0.09 3.03 ± 0.49 > 2.34 2.03 ± 0.27 > 3.34 60 1.80 ± 0.23 > 3.64 > 2.34 > 2.33 3.43 ± 0.01 30 1.51 ± 0.08 1.68 ± 0.28 1.26 ± 0.09 1.81 ± 0.40 1.57 ± 0.05 OLG 60 1.63 ± 0.15 1.69 ± 0.36 1.18 ± 0.10 1.75 ± 0.30 1.66 ± 0.38 1 3 Food and Environmental Virology (2020) 12:180–190 183 between 2.65 and 18.78 mg/mL (Table S1). RNA copy num- bers of norovirus GII.2, GII.4 Den Haag 2006b and GII.12 were below the quantification limit after 30-s contact with OLG-HR (Table 5). The LRVs of GII.10 and GII.14 at 30-s contact time were 1.78 (± 0.13) and 2.44 (± 0.11), respec- tively, which were the highest LRVs among all test materi- als. EtOH-A had a significant virucidal effect only on GII.4 Den Haag 2006b, but was inee ff ctive for the other GII geno - types inactivation (Table 5). The median LRVs of norovirus GII genotypes were 2.41, 0.04, 0.60, 0.52, and 1.27 when exposed to OLG-HR, EtOH, EtOH-A, Base, and OLG for 30-s contact time, respectively (Fig. 2a). At 60-s contact time, these values were 2.37, 0.13, 0.46, 0.67, and 1.16, respectively (Fig. 2b). OLG-HR clearly had the strongest virucidal ee ff ct against tested norovirus GII genotypes, as in the case for norovirus GI genotypes. There was no difference in LRVs between the 30- and 60-s contact times (Fig. 2). There was no correlation between protein concentration of norovirus stool samples and LRVs with each test material after both 30- and 60-s (Table S1 and Fig. S1). The negative control (PBS) virus and protein concen- tration of norovirus GIV.1 were 5.24 (± 0.18) log and 4.29 mg/mL, respectively (Table S1). Against the norovirus GIV.1 genotype, OLG-HR and EtOH-A had the strongest virucidal effect (> 4.24 log ). Also, the mean LRV of noro- virus GIV.1 was higher at the 60-s contact time compared to 30-s when the Base was used. EtOH and OLG were less effective even at the 60-s contact time (Table  5). Discussion In this study, we evaluated the virucidal effect of five anti- septics against eleven genotypes of human norovirus using a culture-independent method. Antiseptics OLG-HR, EtOH, EtOH-A, Base and OLG were used as test materials. There was no influence from the protein concentration of stool samples and residue of test materials for the norovirus log reduction. When OLG-HR was used, LRVs of norovirus Fig. 1 Comparison of virucidal efficacy of test materials against nor - GI.4, GI.6, GI.7, GII.2, GII.4 Den Haag 2006b, GII.12, and ovirus GI genotypes. Log reductions of GI.2, GI.3, GI.4, GI.6, and GIV.1 exceeded the quantification limit at 30-s, and the LRV GI.7 were expressed collectively as box plots for 30- (a) and 60-s (b). The boxes not sharing a common letter differ significantly at p < 0.05 of GI.3 exceeded the quantification limit at 60-s. The LRVs by Steel–Dwass test of norovirus GI.2, GII.10, GII.14 did not exceed the quantifi- cation limit, but the largest LRVs were obtained when OLG- HR was used. OLG-HR was the most effective against noro- Virucidal Efficacy of Test Materials Against Norovirus GII and GIV Genotypes virus GII.2, which emerged in Japan in 2016–2017 season (Nagasawa et al. 2018). Meanwhile, Base and OLG were less The virucidal efficacy of hand sanitizers against norovirus effective compared to OLG-HR. Base had a strong virucidal effect against norovirus GI genotypes, as with OLG-HR, but GII and GIV genotypes was evaluated using the modified RT-qPCR assay (Table 5). The negative control (PBS) nor- was clearly inferior to OLG-HR against norovirus GII and GIV.1. The LRVs of norovirus GI genotypes were between ovirus GII.2, GII.4 Den Haag 2006b, GII.10, GII.12 and GII.14 concentrations were between 3.14 and 3.85 log , 1.18 and 1.81 when OLG was used. EtOH was more effec- tive against norovirus GI genotypes than EtOH-A, but was and the protein concentrations of five GII stool samples were 1 3 184 Food and Environmental Virology (2020) 12:180–190 Table 5 Virucidal efficacy of test materials against norovirus GII and GIV.1 genotypes Test material Reaction time Log reduction [mean ± SD, n = 3] (s) GII.2 GII.4 2006b GII.10 GII.12 GII.14 GIV.1 OLG-HR 30 > 2.85 > 2.71 1.78 ± 0.13 > 2.37 2.44 ± 0.11 > 4.24 60 > 2.85 > 2.71 1.83 ± 0.14 > 2.37 2.63 ± 0.10 > 4.24 EtOH 30 0.16 ± 0.24 0.91 ± 0.12 0.02 ± 0.09 − 0.18 ± 0.19 0.15 ± 0.31 1.86 ± 0.18 60 0.16 ± 0.03 1.13 ± 0.16 0.02 ± 0.15 − 0.04 ± 0.05 0.26 ± 0.23 1.92 ± 0.09 EtOH-A 30 − 0.21 ± 0.06 > 2.71 0.51 ± 0.20 0.27 ± 0.26 0.69 ± 0.34 > 4.24 60 − 0.13 ± 0.03 > 2.71 0.53 ± 0.02 0.16 ± 0.17 0.76 ± 0.08 3.79 ± 0.08 Base 30 0.58 ± 0.09 2.10 ± 0.33 − 0.01 ± 0.17 − 0.05 ± 0.07 0.69 ± 0.16 2.28 ± 0.44 60 0.76 ± 0.15 2.08 ± 0.27 0.06 ± 0.15 0.15 ± 0.05 0.70 ± 0.06 3.81 ± 0.20 OLG 30 0.89 ± 0.09 1.34 ± 0.25 1.42 ± 0.09 0.93 ± 0.29 2.29 ± 0.10 1.61 ± 0.37 60 0.91 ± 0.14 1.12 ± 0.27 1.32 ± 0.13 0.88 ± 0.37 2.28 ± 0.19 1.90 ± 0.35 less effective against norovirus GII genotypes. Overall, it the LRVs obtained by a culture-dependent method (plaque was evident that OLG-HR has the strongest virucidal effects assay) and a culture-independent method using culturable among the five evaluated disinfectants. MNV (Gonzalez-Hernandez et al. 2012). Similar to pre- Costantini et al. showed that a 70% ethanol solution can vious studies, the susceptibility of MNV to ethanol-based slightly disinfect norovirus GII.4 (Costantini et al. 2018). antiseptics was confirmed using both methods (Belliot et al. Another study reported < 0.5 log for human norovirus 2008; Park et al. 2010). MNV was less susceptible to OLG GII.2 and GII.4 by ethanol disinfection (Tung et al. 2013). irrespective of the virus quantification method than other test Because of the complicated culture protocols of human materials. These results indicate that the culture-independent noroviruses (Ettayebi et al. 2016), virucidal effects of dis- method employed in this study may follow the reduction infectants on human norovirus have been evaluated using tendency of viral infectivity. Furthermore, the previously surrogate viruses (Farkas et al. 2010; Hirneisen and Kniel reported virucidal ec ffi acy against norovirus GII.4 is similar 2013; Hoelzer et al. 2013). Unlike human norovirus, ethanol to our test results of norovirus GII.4 Den Haag 2006b and disinfection was effective for the above surrogate viruses therefore, our test results are considered to be reasonable (Cromeans et al. 2014; Kampf et al. 2005; Park et al. 2010). (Costantini et al. 2018). On the other hand, the culture-inde- Our results indicate that unlike feline calicivirus, acidic etha- pendent methods tended to underestimate the MNV LRV nol is only effective against few human norovirus genotypes. compared to the culture-dependent method. Therefore, we Although the reason for this difference is not well under - considered that culture-independent methods can compare stood, feline calicivirus, which causes upper respiratory the relative virucidal efficacy of antiseptics or resistance of disease, probably does not need to withstand environmental genotypes, but not show a true value of LRVs. changes such as strong acidic conditions to establish infec- The results for virucidal effects on norovirus varied tion (Gaskell et al. 2007). However, this study shows that greatly among genogroups and genotypes. As for the ten- results obtained from surrogate viruses do not necessarily dency of difference in resistance by strain, norovirus GII mimic human norovirus, and, therefore, it is not clear how seemed to be more resistant to antiseptics in general than the sensitivity of these surrogate viruses to antiseptics can norovirus GI. In addition, antiseptic resistances were not be compared with that of human norovirus. the same among genogroups; for example, the norovirus Alternatively, culture-independent methods for investi- GI.2 genotype was relatively resistant among norovirus gating the infectivity of viruses have been used to evalu- GII. Park et al. reported that the resistance of norovirus to ate the infectivity of enteric viruses (Sano et al. 2015). The ethanol disinfection differs between genotypes. However, the culture-independent methods are based on RT-qPCR, and mechanism of antiseptic resistance has not yet been clarified RNase or photo-reactive intercalators like PMAxx to dis- (Park et al. 2016). Human norovirus tends to undergo higher tinguish undamaged (likely infectious) viral particles from genome mutation events than DNA-based microorganisms damaged ones (Fraisse et al. 2018; Karim et al. 2015; Park (Bull et al. 2005, 2010; 2012; Sanjuan et al. 2010). Further- et al. 2016; Randazzo et al. 2016). In considering the results more, differences in several nucleotide sequences in open of the culture-independent methods, the correlation with the reading frames (ORF) 2 and ORF3 that encode the major results of culture-dependent methods is very important. In capsid protein (VP1) and minor capsid protein (VP2) may this study, prior to the evaluation of the virucidal efficacy influence disinfection resistance (Park et al. 2016; Rachmadi of these antiseptics against human norovirus, we compared et al. 2018). Thus, the difference in the virucidal efficacy 1 3 Food and Environmental Virology (2020) 12:180–190 185 virucidal effect of OLG-HR against non-enveloped viruses such as feline calicivirus and bacteriophage MS2 has been found to be stronger under basic conditions (data not shown). From these results, the mechanism of action of OLG-HR is expected to be the enhanced protein denaturation action of olanexidine under basic conditions by destroying the capsid structure and exposing the viral genome of human norovirus. EtOH is the most fundamental component of hand sani- tizers. EtOH was effective against norovirus GI.4, GI.6, and GI.7 of GI genotypes, but it was not effective against noro- virus GII genotypes and GIV.1. On the other hand, EtOH-A was effective only against GII.4 Den Haag 2006b and GIV.1. Virucidal effects of acidic, neutral and basic ethanol (EtOH- A, EtOH and Base) were completely different confirming the influence of pH on the sensitivity of viruses to antiseptics. In addition, the pH dependent susceptibility varied among. It is known that the capsid structure of human norovirus is stable between pH 3 and 7, whereas at pH 10 the capsid structure loses its stability (Ausar et al. 2006; Cuellar et al. 2010). The pH of Base was adjusted to be 9.5, and it is possible to expect that the weak basic environment created by Base destabilizes the capsid structure and enhances the virucidal efficacy of olanexidine gluconate and ethanol. In fact, Base had greater virucidal activity against norovirus GI genotypes than EtOH and EtOH-A with efficacy equivalent to OLG-HR. The higher stability of human noroviruses to acidic ethanol may be related to their life cycle to establish infection. This is because human norovirus must withstand dramatic changes in gastrointestinal environmental condi- tions, such as pH 7 in the upper gastrointestinal tract and high acidity in the stomach until it reaches the small intestine at the site of infection. In conclusion, OLG-HR has a stronger virucidal activ- ity at 30- and 60-s contact time for human norovirus than EtOH, EtOH-A, Base, and OLG. OLG-HR has the potential to become the prevention and control tool against human norovirus infection. Fig. 2 Comparison of virucidal efficacy of test materials against Material and Methods norovirus GII genotypes. Log reductions of GII.2, GII.4 Den Haag 2006b, GII.10, GII.12, and GII.14 were expressed collectively as box plots for 30- (a) and 60-s (b). The boxes not sharing a common letter Test Viruses and Cells differ significantly at p < 0.05 by Steel–Dwass test MNV (strain S99) was purchased from Friedrich-Loeffler- of OLG-HR and other antiseptics is thought to be due to Institut (Greifswald island Riems, Germany). RAW264.7 differences in the capsid structure stability by a trivial dif- cells were used as the host of MNV (DS Pharma Biomedical, ference in the amino acid sequence structure, which needs Osaka, Japan). RAW264.7 cells were grown with Dulbecco’s to be addressed in a future study. Modified Eagle’s Medium (DMEM; FUJIFILM Wako Pure The mechanism of action of virucidal efficacy of OLG- Chemical Corporation, Osaka, Japan) containing 10% fetal HR against human norovirus is not well understood, but it bovine serum (Invitrogen, Carlsbad, CA, USA). MNV stocks is known that at relatively high concentrations, olanexidine were inoculated into host cells for 1 h and then cultured for aggregates the cells through a protein-denaturing effect 3 days after the medium change. Three freeze–thaw cycles (Hagi et al. 2015; Sakagami et al. 2000). Furthermore, the were performed and centrifuged (3080×g, 4 °C, 30 min). 1 3 186 Food and Environmental Virology (2020) 12:180–190 After that, the supernatant was collected and ultracentri- Modified RT‑qPCR fuged at 111,000×g at 4 °C for 90 min using a Beckman 50.2 Ti rotor (Beckman Coulter, Inc., Fullerton, CA, USA). Prior to RT-qPCR, the damaged or exposed viral genomes The pellet was suspended in PBS and passed through a filter were removed using previously reported methods (Park et al. with a pore size of 0.45 μm (Millipore, Bedford, MA, USA) 2016; Randazzo et al. 2016). An aliquot (50 μL) of test- and used as test virus solution for the virucidal tests. The material-treated virus solutions was incubated with 56 μL viral genome quantities of the test human norovirus solution of ultrapure water, 8 μL of RNase A (Promega, Madison, used in this study are shown in Table 1. Human noroviruses WI, USA), 2 μL of RNase buffer (Promega, Madison, WI, were obtained from stool specimens collected in Miyagi USA) for 1 h at 37 °C. The reaction was stopped by addition prefecture between 2006 and 2012. Each stool sample was of 4 μL of RNase inhibitor (Invitrogen, Carlsbad, CA, USA), diluted about 10 times with phosphate-buffer saline (PBS, and then the mixture was incubated with 15 μL of PMAxx FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) (Biotium, Hayward, CA, USA), 40 μL of PMA enhancer and centrifuged (4 °C, 10,000×g, 10 min), and the superna- 5 × solution (Biotium, Hayward, CA, USA) and 21 μL of tant was recovered. The supernatant was passed through a ultrapure water for 10 min in darkness and 15 min in visible filter with a pore size of 0.45 μm (Millipore, Bedford, MA, light at room temperature. USA) and used as test virus solution for the virucidal tests. After RNase and PMAxx treatment, MNV RNA of the reaction solutions was extracted using the QIAamp Viral RNA Mini Kit (Qiagen, Hiden, Germany). Reverse Suspension Test transcription was performed using High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Rock - The test materials were olanexidine gluconate hand rub ford IL, USA). The RT reaction mixture was incubated at (OLG-HR), Base component of olanexidine gluconate 25 °C for 10 min, 37 °C for 120 min and 85 °C at 5 min. hand rub (Base), Onaledine Antiseptic Solution 1.5% (OLG, Then, viral RNA was quantified by RT-qPCR as previously Otsuka Pharmaceutical Factory, Inc.), ethanol for disinfec- described (Kitajima et al. 2010) using an ABI PRISM 7500 tion (EtOH, Kenei Pharmaceutical Co., Ltd.) and acidic Fast Real-time PCR System (Applied Biosystems, Foster ethanol containing phosphoric acid (EtOH-A, Saraya Co., City, CA, USA). RT-qPCR mix were performed in 25 μL Ltd.), which is known to be effective for human norovirus reaction volume containing 12.5 μL TaqMan® Gene Expres- surrogate virus (Table 2). Twenty micro-liters of test virus sion Master Mix (Applied Biosystems, Foster City, CA, was mixed with 180 μL of test material (or PBS as a neg- USA), 5 μL cDNA, 400 nM of each primer, and 300 nm ative control) followed by incubation for 30- and 60-s at of TaqMan probe. PCR conditions were initial denaturation room temperature. After the incubation, 50 μL of the reac- at 95 °C for 10 min, 50 cycles of amplification with dena- tion solution was mixed with 450 μL of PBS. After that, turation at 95 °C for 15 s, annealing and extension at 60 °C the gel filtration column (MicroSpin S-400 h Columns; GE for 1 min. Technical replicates for each sample in each RT- Healthcare, Piscataway, NJ, USA) treatment was performed qPCR were set to three (n = 3); the primers and probes used immediately. The gel filtration column was used for the pur - are listed in Table 6. pose of eliminating Olanexidine, which can influence the For human norovirus, the RNA of the reaction solutions subsequent operation (Eterpi et al. 2010). Virucidal tests after RNase and PMAxx treatment was extracted using QIA- were performed in triplicate. cube (Qiagen, Hiden, Germany). Reverse transcription was performed using the iScript Advanced cDNA Synthesis Kit Plaque Assay for RT-qPCR (Bio-Rad, Hercules, CA, USA). The RT reac- tion mixture was incubated at 42 °C for 30 min, and then Plaque assay was conducted as previously reported (Gon- at 85 °C for 5 min to inactivate the enzyme. RT-qPCR for zalez-Hernandez et al. 2012). Host cells on 6-well plates human norovirus was performed using a CFX Connect Real- were inoculated with 500 μL of tenfold dilution series of Time PCR detection system (Bio-Rad, Hercules, CA, USA). each reaction solution and incubated at room temperature RT-qPCR mix were performed in 20 μL reaction volume for 1 h (n = 3). After removal of the reaction solution, an containing 10 μL SsoAdvanced™ Universal Probes Super- agar medium was added and incubated for 2 days at 37 °C. mix (Bio-Rad, Hercules, CA, USA), 5 μL cDNA, 400 nM of After that, 2 mL 0.01% neutral red solution (Sigma-Aldrich, each primer, and 300 nm of TaqMan probe. PCR conditions St Louis, MO, USA) was added and incubated for 4 h at were initial denaturation at 95 °C for 15 min to activate DNA 37 °C. The neutral red solution was removed and the plaques polymerase, 40 cycles of amplification with denaturation at were visually counted. Each assay was repeated three times. 95 °C for 15 s, annealing at 56 °C for 1 min and extension at Log reductions of PFU were calculated, and results were 72 °C for 30 s (Kageyama et al. 2003; Trujillo et al. 2006). expressed as mean ± SD. Technical replicates for each sample in each RT-qPCR were 1 3 Food and Environmental Virology (2020) 12:180–190 187 Table 6 RT-qPCR primers and probes used in this study d e Target virus Primer and probe Seaquence (5′–3′) Polality Location (bp) Length References Murine norovirus MNV-SCCG CAG GAA CGC TCA GCA G + 5028–5046 19 Kitajima et al. (2010) MNV-ASCAG GCC GTC CCC ATT CAG CC − 5137–5156 20 MNV-TPATG AGT GAT GGC GCA + 5062–5076 15 Human norovirus GI COG1FCGY TGG ATGCGNTTY CAT GA + 5291–5110 20 Kageyama et al. (2003) COG1RCTT AGA CGC CAT CAT CAT TYAC − 5354–5375 22 RING1(a)-TPAGA TYG CGA TCY CCT GTC CA − 5021–5340 20 RING1(b)-TPAGA TCG CGG TCT CCT GTC CA − 5021–5340 20 Human norovirus GII COG2FCAR GAR BCNATG TTY AGR TGG + 5003–5028 26 Kageyama et al. (2003) ATGAG COG2RTCG ACG CCA TCT TCA TTC ACA − 5080–5100 21 RING2-AL-TPTGG GAG GGSGAT CGC RATCT + 5048–5067 20 Human norovirus GIV Mon4FTTT GAG TCY ATG TAC AAG TGG ATG + 718–742 25 Trujillo et al. (2006) Mon4RTCG ACG CCA TCT TCA TTC ACA − 795–815 21 Ring4TGG GAG GGG GAT CGC GAT CT + 763–782 20 5′- labeled with 6-carboxyfluorescein (FAM) and 3′-labeled minor groove binder (MGB)-non-fluorescent quencher (NFQ) 5′-labeled with 6-carboxyfluorescein (FAM) and 3′-labeled with Carboxytetramethylrhodamine (TAMRA) 5′-labeled with 6-carboxyfluorescein (FAM) and 3′-labeled with black hole quencher dye (BHQ) Mixed bases in degenerate primers and probes are as follows: Y, C or T; R, A or G; B, not A; N, any Nucleotide position based on MNV-1(GenBank accession no. AY228235), human norovirus GI (GenBank accession no. M87661), human nor- ovirus GII (GenBank accession no. AF145896) and human norovirus GIV (GenBank accession no. AF414426) set to three (n = 3); the primers and probes used are listed in expressed as a box plot. The upper and lower ends of the Table 6. The quantification limit of RT-qPCR of norovirus box and the horizontal line in the box indicate the first and GI, GII, and GIV.1 were 10 copies/well. The PCR efficiency third quantiles and the median value of data, respectively. of norovirus GI, GII, and GIV.1 were 95.78%, 90.31%, and The lower and higher ends of whiskers indicate the mini- 95.02%, respectively. mum and maximum value of the data, respectively. Isolated data points are outliers. Log reduction of each test material Statistics and Data Analysis for Virucidal Tests was analyzed with the Steel–Dwass non-parametric multiple comparison tests following the Kruskal–Wallis test. Data Numerical values of each well of negative control (PBS) analysis was performed using Microsoft Excel 2010, and and test materials obtained by RT-qPCR were taken for a statistical analysis was performed using the statistical soft- common logarithm. For the numerical values of the test ware SAS 9.2 (SAS Institute Japan) and EXSUS 7.7 soft- materials of the three independent tests, log reduction was ware (CAC Excare Corporation). calculated compared to the negative control, and its average values and SD were calculated. When the numerical value of each well was below the limit of detection (10 copies/ Correlation of Protein Concentration of Stool well in this test), the corresponding well was excluded from Samples and LRVs the subsequent calculation and expressed as BDL (below detection limit). If a BDL value was obtained twice or more This experiment was performed to confirm that fecal materi - in three technical triplicates, the numerical value in the test als did not affect the efficacy of test materials in this study. was BDL. If a BDL value was obtained twice or more in The protein concentrations of stool samples were calcu- three independent tests, the RNA copies number of the test lated by measuring the absorbance at 280 nm by NanoDrop was BDL, and the LRV was set to be greater than the maxi- (Thermo Fisher Scientific, Rockford IL, USA). Correlation mum detectable LRV (ex. > 3.5). In the statistical analysis, between protein concentrations of stool samples and the the numerical processing method was changed. That is, if mean of LRVs for each test material were evaluated with the numerical value of each well was BDL, the value of Spearman’s rank correlation coefficient (correlation coef- the corresponding well was 10. The log reduction of each ficient (r ) and p values). The graphs were created using test material of the same genogroup was consolidated and Microsoft Excel 2010, and statistical analysis was performed 1 3 188 Food and Environmental Virology (2020) 12:180–190 Journal of biological chemistry, 281(28), 19478–19488. https :// using the statistical software SAS 9.2 and EXSUS 7.7 doi.org/10.1074/jbc.M6033 13200 . software. Barclay, L., Park, G. W., Vega, E., Hall, A., Parashar, U., & Lopman, B. (2015). Infection control for norovirus. Clinical Microbiol- ogy and Infection, 20(8), 731–740. https://doi.or g/10.1111/1469- Estimating the Eec ff t of Antiseptic Residues 0691.12674 .Infec tion. on Modified RT‑qPCR Reaction Bartsch, S. M., Lopman, B. A., Ozawa, S., Hall, A. J., & Lee, B. Y. (2016). Global economic burden of norovirus gastroenteri- tis. PLoS ONE, 11(4), e0151219. https ://doi.org/10.1371/journ This experiment was performed to confirm that antiseptic al.pone.01512 19. residues did not affect the efficacy of quantification test. Belliot, G., Lavaux, A., Souihel, D., Agnello, D., & Pothier, P. (2008). Twenty micro-liters of PBS was mixed with 180 μL of test Use of murine norovirus as a surrogate to evaluate resistance of human norovirus to disinfectants. Applied and Environmen- material (or PBS as a negative control) at room temperature. tal Microbiology, 74(10), 3315–3318. https ://doi.org/10.1128/ 50 μL of the reaction solution was mixed with 450 μL of AEM.02148 -07. PBS. After MicroSpin S-400 h treatment was performed, Berger, C. N., Sodha, S. V., Shaw, R. K., Griffin, P. M., Pink, D., Hand, added 1/100 volume of stool samples of four genotypes and P., et al. (2010). Fresh fruit and vegetables as vehicles for the trans- mission of human pathogens. Environmental Microbiology, 12(9), carried out the modified RT-qPCR. Technical replicates for 2385–2397. https ://doi.org/10.1111/j.1462-2920.2010.02297 .x. each sample in each RT-qPCR were set to three (n = 3). Boyce, J. M., & Pittet, D. (2002). Guideline for hand hygiene in health- care settings: Recommendations of the healthcare infection con- Acknowledgements We are deeply grateful to Mr. Yoshimitsu Konta trol practices advisory committee and the HICPAC/SHEA/APIC/ at Tohoku University for his technical advice and Ms. Sachi Yamagawa IDSA hand hygiene task force. Infection Control & Hospital Epi- at Otsuka Pharmaceutical Factory, Inc. for technical assistance with demiology, 23(S12), S3–S40. https ://doi.org/10.1086/50316 4. the experiments. Bull, R. A., Eden, J.-S., Luciani, F., McElroy, K., Rawlinson, W. D., & White, P. A. (2012). Contribution of intra- and interhost dynamics to norovirus evolution. Journal of Virology, 86(6), 3219–3229. Compliance with Ethical Standards https ://doi.org/10.1128/JVI.06712 -11. Bull, R. A., Eden, J.-S., Rawlinson, W. D., & White, P. A. (2010). Conflict of interest This research is a collaborative study between Rapid evolution of pandemic noroviruses of the GII.4 lineage. Tohoku University and Otsuka Pharmaceutical Factory, Inc., and is PLoS Pathogens, 6(3), e1000831. https ://doi.org/10.1371/journ sponsored by Otsuka Pharmaceutical Factory, Inc. This study may lead al.ppat.10008 31. to the development of products that may be licensed to Otsuka Phar- Bull, R. A., Hansman, G. S., Clancy, L. E., Tanaka, M. M., Rawlinson, maceutical Factory, Inc. After assessment by Tohoku University, this W. D., & White, P. A. (2005). Norovirus recombination in ORF1/ study has been found to have no conflicts of interest. KI, AH and YI ORF2 overlap. Emerging Infectious Diseases, 11(7), 1079–1085. are employees of Otsuka Pharmaceutical Factory, Inc. However, the https ://doi.org/10.3201/eid11 07.04127 3. company had no control over the interpretation of the results or the Campos, C. J. A., & Lees, D. N. (2014). Environmental transmission writing and publication of this study. of human noroviruses in shellfish waters. 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Publisher’s Note Springer Nature remains neutral with regard to https ://doi.org/10.1128/AEM.03802 -14. jurisdictional claims in published maps and institutional affiliations. Sickbert-Bennett, E. E., Weber, D. J., Gergen-Teague, M. F., Sobsey, M. D., Samsa, G. P., & Rutala, W. A. (2005). Comparative efficacy of hand hygiene agents in the reduction of bacteria and viruses. 1 3 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Food and Environmental Virology Springer Journals

Virucidal Efficacy of Olanexidine Gluconate as a Hand Antiseptic Against Human Norovirus

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10.1007/s12560-020-09422-4
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Abstract

Human noroviruses are the major cause of non-bacterial acute gastroenteritis worldwide. Since no therapeutic agent has been proven to prevent human norovirus infection yet, preventive healthcare interventions to block the infection routes play an important role in infection control. One of the possible infection routes of human noroviruses are through contaminated hands, but no hand antiseptics have been proven effective. Olanexidine gluconate is a new biguanide compound that has already been approved for sale as an antiseptic for the surgical field in Japan. A new hand antiseptic was developed using olanexidine gluconate in this study, and its virucidal efficacy against human noroviruses was evaluated using modified RT- qPCR that can account for genome derived from intact viruses using RNase A and photo-reactive intercalators. We tested the virucidal efficacy of five materials; two olanexidine gluconate antiseptics (hand rub formulation and surgical field formula- tion), two kinds of ethanol solutions at different pH (approx. 3 or 7), and a base component of olanexidine gluconate hand rub formulation against 11 human norovirus genotypes by culture-independent methods. The infectivity of murine norovirus (MNV), a surrogate for human norovirus, was significantly reduced after use of the antiseptics. The olanexidine gluconate hand rub demonstrated the strongest virucidal efficacy against human norovirus among the five tested materials. This study showed that olanexidine gluconate has the potential to become a strong tool for the prevention of human norovirus infection. Keywords Human norovirus · Inactivation · Olanexidine · Antiseptic · RT-qPCR Introduction (Adams et al. 2016). Of the ten genogroups of norovirus, genogroups I (GI), GII, GIV, GVIII, and GIX can infect Noroviruses are a non-enveloped and single-stranded posi- humans, and GIII, GV, GVII, and GX can infect bovine, tive-sense RNA virus belonging to the family Caliciviridae, mice, dogs, and bats, respectively, (Chhabra et al. 2019). and its particle is an icosahedron with a diameter of 38 nm Infectivity of human norovirus is considered to be very strong; fewer than ten viral particles have been reported adequate to establish infection (Feng et al. 2011). Human Electronic supplementary material The online version of this noroviruses cause acute gastroenteritis in medical facilities, article (https ://doi.org/10.1007/s1256 0-020-09422 -4) contains schools and restaurants, and the economic burden of the ill- supplementary material, which is available to authorized users. ness is $4.2 billion and $60.3 billion in direct medical and indirect societal costs per year, respectively (Bartsch et al. * Daisuke Sano daisuke.sano.e1@tohoku.ac.jp 2016). Varied infection vehicles of human norovirus have been reported, including shellfish (Campos and Lees 2014), Naruto Research Institute, Research and Development fruits (Berger et al. 2010) and other foods such as dried laver Center, Otsuka Pharmaceutical Factory, Inc., Naruto, seaweed (Kusumi et al. 2017). Human noroviruses grow in Tokushima 772-8601, Japan small intestinal epithelial cells after a latency period of 12 Department of Frontier Science for Advanced Environment, to 48 h and cause symptoms such as abdominal pain, diar- Graduate School of Environmental Studies, Tohoku University, Aoba 6-6-06, Aramaki, Aoba-ku, Sendai, rhea, nausea and vomiting (Dolin et al. 1972). Usually the Miyagi 980-8579, Japan symptoms disappear within 2 days, but they may be severe Department of Civil and Environmental Engineering, depending on the physical strength and hygienic condition of Graduate School of Engineering, Tohoku University, Aoba patients (Fischer Walker et al. 2012; Liu et al. 2012). Human 6-6-06, Aramaki, Aoba-ku, Sendai, Miyagi 980-8579, Japan Vol:.(1234567890) 1 3 Food and Environmental Virology (2020) 12:180–190 181 norovirus particles released from the feces of infected people improved version of OLG, ethanol was added to provide are resistant to drying and heat, and maintain infectivity in quick drying, and the pH was adjusted to be alkaline to natural environments, which poses a risk of secondary infec- enhance the capsid denaturation effect. The virucidal effi- tion (Barclay et al. 2015; Lopman et al. 2012; Robilotti et al. cacy of five antiseptics including OLG-HR against eleven 2015). Since no therapeutic agent or vaccine has been proven different genotypes of human norovirus was evaluated by a effective against human noroviruses, hygienic interventions, culture-independent method (RT-qPCR) using RNase A and including hand washing, are regarded important for control photo-reactive intercalator PMAxx (propidium monoazide) of the infection (Barclay et al. 2015; Park et al. 2014; Park pretreatment step. The virucidal effects of OLG-HR were and Sobsey 2011) (Table 1). compared with those of ethanol for disinfection (EtOH), Olanexidine gluconate is a novel antimicrobial compound acidically adjusted ethanol (pH 3) for disinfection (EtOH- developed by Otsuka Pharmaceutical Factory, Inc. and has A), base ingredient excluding olanexidine gluconate from been used in Japan clinically for preoperative skin prepa- OLG-HR (Base), and OLG (Table 2). EtOH was used as a ration [Product name: Olanedine Antiseptic Solution 1.5% test antiseptic because hand sanitizers containing ethanol has (OLG)] (Hagi et al. 2015; Inoue et al. 2015; Nakata et al. been recommended for the disinfection of hands by the Cent- 2017). The virucidal efficacy of OLG against feline calici- ers for Disease Control and Prevention (CDC) and World virus at 60-s and 10 min were 0.60, and 2.85, respectively Health Organization (WHO) (Boyce and Pittet 2002; WHO (unpublished data). Because European Committee for Stand- 2009). EtOH-A was used because of its high inactivation ardization states that “the product shall demonstrate at least efficiency against surrogate viruses, although its efficacy for a decimal log reduction of 4 in virus titre” (EN 14476 2013), human noroviruses were unknown (Park et al. 2010). The the virucidal activity of OLG against a non-enveloped virus disinfection efficiency of OLG-HR and other four antisep - is not enough as well as other biguanide compounds (Iwa- tics against multiple genotypes within GI, GII and GIV of sawa et al. 2012; Sickbert-Bennett et al. 2005). The mode of noroviruses were compared. action of OLG is similar to that of chlorhexidine gluconate, a biguanide antiseptic, but OLG has also been confirmed to have a stronger protein denaturation effect than chlorhex- Results idine gluconate (Hagi et al. 2015). The objective of this study is to develop a new olanex- Virucidal Efficacy of Test Materials Against Murine idine-containing formulation for hand hygiene (olanexidine Norovirus by Modified RT‑qPCR and Plaque Assay gluconate hand rub; OLG-HR) in anticipation of the pro- tein denaturation activity of OLG for destroying the cap- Since we have no simple in vitro culture method for human sid protein of human noroviruses. In OLG-HR, which is an noroviruses, it is difficult to evaluate the correlation between the value of RT-qPCR and the virus infectivity. Therefore, we compared the results from the modified RT-qPCR and Table 1 Genotype and viral titer of norovirus positive stool samples the plaque assay using murine norovirus (MNV) (Table 3). in this study The virucidal efficiency of each test material was similar Genogroup Genotype Quantity (Log except for OLG. The log reduction values (LRVs, ± SD) RNA copies/well) of OLG at 30- and 60-s contact time evaluated using the Human norovirus GI GI.2 4.45 GI.3 5.75 Table 2 Details of antiseptics evaluated in this study GI.4 4.85 Test material Active ingredient pH GI.6 4.07 Name Concentration of GI.7 5.75 olanexidine gluco- Human norovirus GII GII.2 4.52 nate (%) GII.4 Den Haag 4.82 2006b OLG-HR Olanexidine gluco- 1.5 9.5 GII.10 4.17 nate/ethanol (70%) GII.12 4.56 EtOH Ethanol (76.9– – Approx. 7 81.4%) GII.14 4.29 EtOH-A Ethanol (76.9– – Approx. 3 Human norovirus GIV GIV.1 6.16 81.4%) This value was RNA copies number of 100-fold diluted solution of Base Ethanol (70%) – 9.5 test virus solution. This measurement was carried out prior to the OLG Olanexidine gluco- 1.5 5 virucidal activity test in order to confirm the concentration of the nate stool samples 1 3 182 Food and Environmental Virology (2020) 12:180–190 Table 3 Virucidal efficacy of Test material Reaction Log reduction of modified RT-qPCR Log reduction by plaque assay test materials against murine time (s) [log copies/well, mean ± SD, n = 3] [log PFU/mL, mean ± SD, 10 10 norovirus S99 by modified n = 3] RT-qPCR and plaque assay OLG-HR 30 3.92 ± 0.21 > 4.58 60 4.04 ± 0.18 > 4.58 EtOH 30 4.00 ± 0.25 > 4.58 60 3.96 ± 0.17 > 4.58 EtOH-A 30 3.90 ± 0.16 > 4.58 60 4.05 ± 0.22 > 4.58 Base 30 4.05 ± 0.21 > 4.58 60 3.96 ± 0.20 > 4.58 OLG 30 1.90 ± 0.14 2.35 ± 0.22 60 1.83 ± 0.42 2.73 ± 0.80 plaque assay were 2.35 (± 0.22) and 2.73 (± 0.80), and those with OLG-HR were 30- and 60-s, respectively, which using the modified RT-qPCR assay were 1.90 (± 0.14) and were similar to the results of Base. The mean LRVs of 1.83 (± 0.42), respectively. Log reduction obtainable by the EtOH against norovirus GI.4, GI.6, and GI.7 were > 2.34 other four test materials containing ethanol were between log (higher than the quantification limit), > 2.33 log , 10 10 3.90 and 4.04 at both contact times when modified RT-qPCR and 3.20 (± 0.36) log at 30-s, whereas those for noro- assay was used, and those evaluated by the plaque assay virus GI.2 and GI.3 were 0.92 (± 0.31) and 0.10 (± 0.03) were > 4.58. log even after 60-s. EtOH-A was not effective against norovirus GI.3, and the mean LRVs against the 4 other Virucidal Efficacy of Test Materials Against Norovirus GI genotypes were between 1.11 and 1.81 l og at 60-s GI Genotypes (Table 4). The median LRVs of norovirus GI genotypes at 30-s contact time using OLG-HR, EtOH, EtOH-A, Base, The virucidal efficacy of test materials against norovirus and OLG were 2.34, 2.08, 0.74, 2.34, and 1.52 (Fig. 1a), GI genotypes was evaluated using the modified RT-qPCR and the 60-s contact time medians were 2.34, 2.33, 1.25, assay. The RNA copy numbers (log RNA copies/well) of 2.34, and 1.50, respectively (Fig. 1b). The median LRV norovirus GI.2, GI.3, GI.4, GI.6, and GI.7 in PBS (nega- of norovirus GI genotypes was significantly higher when tive control) were between 3.33 to 4.64, and the protein OLG-HR and Base were used at both contact times. The concentrations of five GI stool samples were between 2.19 difference between OLG-HR and Base was not significant and 14.61 mg/mL (Table S1). When OLG-HR was used, at both contact times (Fig. 1). There was no correlation the mean LRVs of norovirus GI.4, GI.6, and GI.7 were between protein concentration of norovirus stool sam- higher than the quantification limit at the 30-s contact ples and LRVs for each test material both 30- and 60-s time (Table 4). The mean LRVs of norovirus GI.2 were (Table S1 and Fig. S1). 1.76 (± 0.18) and 1.70 (± 0.20) when the contact times Table 4 Virucidal efficacy of Test material Reaction Log reduction [mean ± SD, n = 3] test materials against norovirus time (s) GI genotypes GI.2 GI.3 GI.4 GI.6 GI.7 OLG-HR 30 1.76 ± 0.18 3.28 ± 0.54 > 2.34 > 2.33 > 3.34 60 1.70 ± 0.20 > 3.64 > 2.34 > 2.33 > 3.34 EtOH 30 0.72 ± 0.16 0.01 ± 0.25 > 2.34 > 2.33 3.20 ± 0.36 60 0.92 ± 0.31 0.10 ± 0.03 > 2.34 > 2.33 > 3.34 EtOH-A 30 0.99 ± 0.10 0.59 ± 0.07 0.38 ± 0.21 1.24 ± 0.17 0.80 ± 0.25 60 1.16 ± 0.12 0.66 ± 0.02 1.11 ± 0.03 1.63 ± 0.16 1.81 ± 0.33 Base 30 1.75 ± 0.09 3.03 ± 0.49 > 2.34 2.03 ± 0.27 > 3.34 60 1.80 ± 0.23 > 3.64 > 2.34 > 2.33 3.43 ± 0.01 30 1.51 ± 0.08 1.68 ± 0.28 1.26 ± 0.09 1.81 ± 0.40 1.57 ± 0.05 OLG 60 1.63 ± 0.15 1.69 ± 0.36 1.18 ± 0.10 1.75 ± 0.30 1.66 ± 0.38 1 3 Food and Environmental Virology (2020) 12:180–190 183 between 2.65 and 18.78 mg/mL (Table S1). RNA copy num- bers of norovirus GII.2, GII.4 Den Haag 2006b and GII.12 were below the quantification limit after 30-s contact with OLG-HR (Table 5). The LRVs of GII.10 and GII.14 at 30-s contact time were 1.78 (± 0.13) and 2.44 (± 0.11), respec- tively, which were the highest LRVs among all test materi- als. EtOH-A had a significant virucidal effect only on GII.4 Den Haag 2006b, but was inee ff ctive for the other GII geno - types inactivation (Table 5). The median LRVs of norovirus GII genotypes were 2.41, 0.04, 0.60, 0.52, and 1.27 when exposed to OLG-HR, EtOH, EtOH-A, Base, and OLG for 30-s contact time, respectively (Fig. 2a). At 60-s contact time, these values were 2.37, 0.13, 0.46, 0.67, and 1.16, respectively (Fig. 2b). OLG-HR clearly had the strongest virucidal ee ff ct against tested norovirus GII genotypes, as in the case for norovirus GI genotypes. There was no difference in LRVs between the 30- and 60-s contact times (Fig. 2). There was no correlation between protein concentration of norovirus stool samples and LRVs with each test material after both 30- and 60-s (Table S1 and Fig. S1). The negative control (PBS) virus and protein concen- tration of norovirus GIV.1 were 5.24 (± 0.18) log and 4.29 mg/mL, respectively (Table S1). Against the norovirus GIV.1 genotype, OLG-HR and EtOH-A had the strongest virucidal effect (> 4.24 log ). Also, the mean LRV of noro- virus GIV.1 was higher at the 60-s contact time compared to 30-s when the Base was used. EtOH and OLG were less effective even at the 60-s contact time (Table  5). Discussion In this study, we evaluated the virucidal effect of five anti- septics against eleven genotypes of human norovirus using a culture-independent method. Antiseptics OLG-HR, EtOH, EtOH-A, Base and OLG were used as test materials. There was no influence from the protein concentration of stool samples and residue of test materials for the norovirus log reduction. When OLG-HR was used, LRVs of norovirus Fig. 1 Comparison of virucidal efficacy of test materials against nor - GI.4, GI.6, GI.7, GII.2, GII.4 Den Haag 2006b, GII.12, and ovirus GI genotypes. Log reductions of GI.2, GI.3, GI.4, GI.6, and GIV.1 exceeded the quantification limit at 30-s, and the LRV GI.7 were expressed collectively as box plots for 30- (a) and 60-s (b). The boxes not sharing a common letter differ significantly at p < 0.05 of GI.3 exceeded the quantification limit at 60-s. The LRVs by Steel–Dwass test of norovirus GI.2, GII.10, GII.14 did not exceed the quantifi- cation limit, but the largest LRVs were obtained when OLG- HR was used. OLG-HR was the most effective against noro- Virucidal Efficacy of Test Materials Against Norovirus GII and GIV Genotypes virus GII.2, which emerged in Japan in 2016–2017 season (Nagasawa et al. 2018). Meanwhile, Base and OLG were less The virucidal efficacy of hand sanitizers against norovirus effective compared to OLG-HR. Base had a strong virucidal effect against norovirus GI genotypes, as with OLG-HR, but GII and GIV genotypes was evaluated using the modified RT-qPCR assay (Table 5). The negative control (PBS) nor- was clearly inferior to OLG-HR against norovirus GII and GIV.1. The LRVs of norovirus GI genotypes were between ovirus GII.2, GII.4 Den Haag 2006b, GII.10, GII.12 and GII.14 concentrations were between 3.14 and 3.85 log , 1.18 and 1.81 when OLG was used. EtOH was more effec- tive against norovirus GI genotypes than EtOH-A, but was and the protein concentrations of five GII stool samples were 1 3 184 Food and Environmental Virology (2020) 12:180–190 Table 5 Virucidal efficacy of test materials against norovirus GII and GIV.1 genotypes Test material Reaction time Log reduction [mean ± SD, n = 3] (s) GII.2 GII.4 2006b GII.10 GII.12 GII.14 GIV.1 OLG-HR 30 > 2.85 > 2.71 1.78 ± 0.13 > 2.37 2.44 ± 0.11 > 4.24 60 > 2.85 > 2.71 1.83 ± 0.14 > 2.37 2.63 ± 0.10 > 4.24 EtOH 30 0.16 ± 0.24 0.91 ± 0.12 0.02 ± 0.09 − 0.18 ± 0.19 0.15 ± 0.31 1.86 ± 0.18 60 0.16 ± 0.03 1.13 ± 0.16 0.02 ± 0.15 − 0.04 ± 0.05 0.26 ± 0.23 1.92 ± 0.09 EtOH-A 30 − 0.21 ± 0.06 > 2.71 0.51 ± 0.20 0.27 ± 0.26 0.69 ± 0.34 > 4.24 60 − 0.13 ± 0.03 > 2.71 0.53 ± 0.02 0.16 ± 0.17 0.76 ± 0.08 3.79 ± 0.08 Base 30 0.58 ± 0.09 2.10 ± 0.33 − 0.01 ± 0.17 − 0.05 ± 0.07 0.69 ± 0.16 2.28 ± 0.44 60 0.76 ± 0.15 2.08 ± 0.27 0.06 ± 0.15 0.15 ± 0.05 0.70 ± 0.06 3.81 ± 0.20 OLG 30 0.89 ± 0.09 1.34 ± 0.25 1.42 ± 0.09 0.93 ± 0.29 2.29 ± 0.10 1.61 ± 0.37 60 0.91 ± 0.14 1.12 ± 0.27 1.32 ± 0.13 0.88 ± 0.37 2.28 ± 0.19 1.90 ± 0.35 less effective against norovirus GII genotypes. Overall, it the LRVs obtained by a culture-dependent method (plaque was evident that OLG-HR has the strongest virucidal effects assay) and a culture-independent method using culturable among the five evaluated disinfectants. MNV (Gonzalez-Hernandez et al. 2012). Similar to pre- Costantini et al. showed that a 70% ethanol solution can vious studies, the susceptibility of MNV to ethanol-based slightly disinfect norovirus GII.4 (Costantini et al. 2018). antiseptics was confirmed using both methods (Belliot et al. Another study reported < 0.5 log for human norovirus 2008; Park et al. 2010). MNV was less susceptible to OLG GII.2 and GII.4 by ethanol disinfection (Tung et al. 2013). irrespective of the virus quantification method than other test Because of the complicated culture protocols of human materials. These results indicate that the culture-independent noroviruses (Ettayebi et al. 2016), virucidal effects of dis- method employed in this study may follow the reduction infectants on human norovirus have been evaluated using tendency of viral infectivity. Furthermore, the previously surrogate viruses (Farkas et al. 2010; Hirneisen and Kniel reported virucidal ec ffi acy against norovirus GII.4 is similar 2013; Hoelzer et al. 2013). Unlike human norovirus, ethanol to our test results of norovirus GII.4 Den Haag 2006b and disinfection was effective for the above surrogate viruses therefore, our test results are considered to be reasonable (Cromeans et al. 2014; Kampf et al. 2005; Park et al. 2010). (Costantini et al. 2018). On the other hand, the culture-inde- Our results indicate that unlike feline calicivirus, acidic etha- pendent methods tended to underestimate the MNV LRV nol is only effective against few human norovirus genotypes. compared to the culture-dependent method. Therefore, we Although the reason for this difference is not well under - considered that culture-independent methods can compare stood, feline calicivirus, which causes upper respiratory the relative virucidal efficacy of antiseptics or resistance of disease, probably does not need to withstand environmental genotypes, but not show a true value of LRVs. changes such as strong acidic conditions to establish infec- The results for virucidal effects on norovirus varied tion (Gaskell et al. 2007). However, this study shows that greatly among genogroups and genotypes. As for the ten- results obtained from surrogate viruses do not necessarily dency of difference in resistance by strain, norovirus GII mimic human norovirus, and, therefore, it is not clear how seemed to be more resistant to antiseptics in general than the sensitivity of these surrogate viruses to antiseptics can norovirus GI. In addition, antiseptic resistances were not be compared with that of human norovirus. the same among genogroups; for example, the norovirus Alternatively, culture-independent methods for investi- GI.2 genotype was relatively resistant among norovirus gating the infectivity of viruses have been used to evalu- GII. Park et al. reported that the resistance of norovirus to ate the infectivity of enteric viruses (Sano et al. 2015). The ethanol disinfection differs between genotypes. However, the culture-independent methods are based on RT-qPCR, and mechanism of antiseptic resistance has not yet been clarified RNase or photo-reactive intercalators like PMAxx to dis- (Park et al. 2016). Human norovirus tends to undergo higher tinguish undamaged (likely infectious) viral particles from genome mutation events than DNA-based microorganisms damaged ones (Fraisse et al. 2018; Karim et al. 2015; Park (Bull et al. 2005, 2010; 2012; Sanjuan et al. 2010). Further- et al. 2016; Randazzo et al. 2016). In considering the results more, differences in several nucleotide sequences in open of the culture-independent methods, the correlation with the reading frames (ORF) 2 and ORF3 that encode the major results of culture-dependent methods is very important. In capsid protein (VP1) and minor capsid protein (VP2) may this study, prior to the evaluation of the virucidal efficacy influence disinfection resistance (Park et al. 2016; Rachmadi of these antiseptics against human norovirus, we compared et al. 2018). Thus, the difference in the virucidal efficacy 1 3 Food and Environmental Virology (2020) 12:180–190 185 virucidal effect of OLG-HR against non-enveloped viruses such as feline calicivirus and bacteriophage MS2 has been found to be stronger under basic conditions (data not shown). From these results, the mechanism of action of OLG-HR is expected to be the enhanced protein denaturation action of olanexidine under basic conditions by destroying the capsid structure and exposing the viral genome of human norovirus. EtOH is the most fundamental component of hand sani- tizers. EtOH was effective against norovirus GI.4, GI.6, and GI.7 of GI genotypes, but it was not effective against noro- virus GII genotypes and GIV.1. On the other hand, EtOH-A was effective only against GII.4 Den Haag 2006b and GIV.1. Virucidal effects of acidic, neutral and basic ethanol (EtOH- A, EtOH and Base) were completely different confirming the influence of pH on the sensitivity of viruses to antiseptics. In addition, the pH dependent susceptibility varied among. It is known that the capsid structure of human norovirus is stable between pH 3 and 7, whereas at pH 10 the capsid structure loses its stability (Ausar et al. 2006; Cuellar et al. 2010). The pH of Base was adjusted to be 9.5, and it is possible to expect that the weak basic environment created by Base destabilizes the capsid structure and enhances the virucidal efficacy of olanexidine gluconate and ethanol. In fact, Base had greater virucidal activity against norovirus GI genotypes than EtOH and EtOH-A with efficacy equivalent to OLG-HR. The higher stability of human noroviruses to acidic ethanol may be related to their life cycle to establish infection. This is because human norovirus must withstand dramatic changes in gastrointestinal environmental condi- tions, such as pH 7 in the upper gastrointestinal tract and high acidity in the stomach until it reaches the small intestine at the site of infection. In conclusion, OLG-HR has a stronger virucidal activ- ity at 30- and 60-s contact time for human norovirus than EtOH, EtOH-A, Base, and OLG. OLG-HR has the potential to become the prevention and control tool against human norovirus infection. Fig. 2 Comparison of virucidal efficacy of test materials against Material and Methods norovirus GII genotypes. Log reductions of GII.2, GII.4 Den Haag 2006b, GII.10, GII.12, and GII.14 were expressed collectively as box plots for 30- (a) and 60-s (b). The boxes not sharing a common letter Test Viruses and Cells differ significantly at p < 0.05 by Steel–Dwass test MNV (strain S99) was purchased from Friedrich-Loeffler- of OLG-HR and other antiseptics is thought to be due to Institut (Greifswald island Riems, Germany). RAW264.7 differences in the capsid structure stability by a trivial dif- cells were used as the host of MNV (DS Pharma Biomedical, ference in the amino acid sequence structure, which needs Osaka, Japan). RAW264.7 cells were grown with Dulbecco’s to be addressed in a future study. Modified Eagle’s Medium (DMEM; FUJIFILM Wako Pure The mechanism of action of virucidal efficacy of OLG- Chemical Corporation, Osaka, Japan) containing 10% fetal HR against human norovirus is not well understood, but it bovine serum (Invitrogen, Carlsbad, CA, USA). MNV stocks is known that at relatively high concentrations, olanexidine were inoculated into host cells for 1 h and then cultured for aggregates the cells through a protein-denaturing effect 3 days after the medium change. Three freeze–thaw cycles (Hagi et al. 2015; Sakagami et al. 2000). Furthermore, the were performed and centrifuged (3080×g, 4 °C, 30 min). 1 3 186 Food and Environmental Virology (2020) 12:180–190 After that, the supernatant was collected and ultracentri- Modified RT‑qPCR fuged at 111,000×g at 4 °C for 90 min using a Beckman 50.2 Ti rotor (Beckman Coulter, Inc., Fullerton, CA, USA). Prior to RT-qPCR, the damaged or exposed viral genomes The pellet was suspended in PBS and passed through a filter were removed using previously reported methods (Park et al. with a pore size of 0.45 μm (Millipore, Bedford, MA, USA) 2016; Randazzo et al. 2016). An aliquot (50 μL) of test- and used as test virus solution for the virucidal tests. The material-treated virus solutions was incubated with 56 μL viral genome quantities of the test human norovirus solution of ultrapure water, 8 μL of RNase A (Promega, Madison, used in this study are shown in Table 1. Human noroviruses WI, USA), 2 μL of RNase buffer (Promega, Madison, WI, were obtained from stool specimens collected in Miyagi USA) for 1 h at 37 °C. The reaction was stopped by addition prefecture between 2006 and 2012. Each stool sample was of 4 μL of RNase inhibitor (Invitrogen, Carlsbad, CA, USA), diluted about 10 times with phosphate-buffer saline (PBS, and then the mixture was incubated with 15 μL of PMAxx FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) (Biotium, Hayward, CA, USA), 40 μL of PMA enhancer and centrifuged (4 °C, 10,000×g, 10 min), and the superna- 5 × solution (Biotium, Hayward, CA, USA) and 21 μL of tant was recovered. The supernatant was passed through a ultrapure water for 10 min in darkness and 15 min in visible filter with a pore size of 0.45 μm (Millipore, Bedford, MA, light at room temperature. USA) and used as test virus solution for the virucidal tests. After RNase and PMAxx treatment, MNV RNA of the reaction solutions was extracted using the QIAamp Viral RNA Mini Kit (Qiagen, Hiden, Germany). Reverse Suspension Test transcription was performed using High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Rock - The test materials were olanexidine gluconate hand rub ford IL, USA). The RT reaction mixture was incubated at (OLG-HR), Base component of olanexidine gluconate 25 °C for 10 min, 37 °C for 120 min and 85 °C at 5 min. hand rub (Base), Onaledine Antiseptic Solution 1.5% (OLG, Then, viral RNA was quantified by RT-qPCR as previously Otsuka Pharmaceutical Factory, Inc.), ethanol for disinfec- described (Kitajima et al. 2010) using an ABI PRISM 7500 tion (EtOH, Kenei Pharmaceutical Co., Ltd.) and acidic Fast Real-time PCR System (Applied Biosystems, Foster ethanol containing phosphoric acid (EtOH-A, Saraya Co., City, CA, USA). RT-qPCR mix were performed in 25 μL Ltd.), which is known to be effective for human norovirus reaction volume containing 12.5 μL TaqMan® Gene Expres- surrogate virus (Table 2). Twenty micro-liters of test virus sion Master Mix (Applied Biosystems, Foster City, CA, was mixed with 180 μL of test material (or PBS as a neg- USA), 5 μL cDNA, 400 nM of each primer, and 300 nm ative control) followed by incubation for 30- and 60-s at of TaqMan probe. PCR conditions were initial denaturation room temperature. After the incubation, 50 μL of the reac- at 95 °C for 10 min, 50 cycles of amplification with dena- tion solution was mixed with 450 μL of PBS. After that, turation at 95 °C for 15 s, annealing and extension at 60 °C the gel filtration column (MicroSpin S-400 h Columns; GE for 1 min. Technical replicates for each sample in each RT- Healthcare, Piscataway, NJ, USA) treatment was performed qPCR were set to three (n = 3); the primers and probes used immediately. The gel filtration column was used for the pur - are listed in Table 6. pose of eliminating Olanexidine, which can influence the For human norovirus, the RNA of the reaction solutions subsequent operation (Eterpi et al. 2010). Virucidal tests after RNase and PMAxx treatment was extracted using QIA- were performed in triplicate. cube (Qiagen, Hiden, Germany). Reverse transcription was performed using the iScript Advanced cDNA Synthesis Kit Plaque Assay for RT-qPCR (Bio-Rad, Hercules, CA, USA). The RT reac- tion mixture was incubated at 42 °C for 30 min, and then Plaque assay was conducted as previously reported (Gon- at 85 °C for 5 min to inactivate the enzyme. RT-qPCR for zalez-Hernandez et al. 2012). Host cells on 6-well plates human norovirus was performed using a CFX Connect Real- were inoculated with 500 μL of tenfold dilution series of Time PCR detection system (Bio-Rad, Hercules, CA, USA). each reaction solution and incubated at room temperature RT-qPCR mix were performed in 20 μL reaction volume for 1 h (n = 3). After removal of the reaction solution, an containing 10 μL SsoAdvanced™ Universal Probes Super- agar medium was added and incubated for 2 days at 37 °C. mix (Bio-Rad, Hercules, CA, USA), 5 μL cDNA, 400 nM of After that, 2 mL 0.01% neutral red solution (Sigma-Aldrich, each primer, and 300 nm of TaqMan probe. PCR conditions St Louis, MO, USA) was added and incubated for 4 h at were initial denaturation at 95 °C for 15 min to activate DNA 37 °C. The neutral red solution was removed and the plaques polymerase, 40 cycles of amplification with denaturation at were visually counted. Each assay was repeated three times. 95 °C for 15 s, annealing at 56 °C for 1 min and extension at Log reductions of PFU were calculated, and results were 72 °C for 30 s (Kageyama et al. 2003; Trujillo et al. 2006). expressed as mean ± SD. Technical replicates for each sample in each RT-qPCR were 1 3 Food and Environmental Virology (2020) 12:180–190 187 Table 6 RT-qPCR primers and probes used in this study d e Target virus Primer and probe Seaquence (5′–3′) Polality Location (bp) Length References Murine norovirus MNV-SCCG CAG GAA CGC TCA GCA G + 5028–5046 19 Kitajima et al. (2010) MNV-ASCAG GCC GTC CCC ATT CAG CC − 5137–5156 20 MNV-TPATG AGT GAT GGC GCA + 5062–5076 15 Human norovirus GI COG1FCGY TGG ATGCGNTTY CAT GA + 5291–5110 20 Kageyama et al. (2003) COG1RCTT AGA CGC CAT CAT CAT TYAC − 5354–5375 22 RING1(a)-TPAGA TYG CGA TCY CCT GTC CA − 5021–5340 20 RING1(b)-TPAGA TCG CGG TCT CCT GTC CA − 5021–5340 20 Human norovirus GII COG2FCAR GAR BCNATG TTY AGR TGG + 5003–5028 26 Kageyama et al. (2003) ATGAG COG2RTCG ACG CCA TCT TCA TTC ACA − 5080–5100 21 RING2-AL-TPTGG GAG GGSGAT CGC RATCT + 5048–5067 20 Human norovirus GIV Mon4FTTT GAG TCY ATG TAC AAG TGG ATG + 718–742 25 Trujillo et al. (2006) Mon4RTCG ACG CCA TCT TCA TTC ACA − 795–815 21 Ring4TGG GAG GGG GAT CGC GAT CT + 763–782 20 5′- labeled with 6-carboxyfluorescein (FAM) and 3′-labeled minor groove binder (MGB)-non-fluorescent quencher (NFQ) 5′-labeled with 6-carboxyfluorescein (FAM) and 3′-labeled with Carboxytetramethylrhodamine (TAMRA) 5′-labeled with 6-carboxyfluorescein (FAM) and 3′-labeled with black hole quencher dye (BHQ) Mixed bases in degenerate primers and probes are as follows: Y, C or T; R, A or G; B, not A; N, any Nucleotide position based on MNV-1(GenBank accession no. AY228235), human norovirus GI (GenBank accession no. M87661), human nor- ovirus GII (GenBank accession no. AF145896) and human norovirus GIV (GenBank accession no. AF414426) set to three (n = 3); the primers and probes used are listed in expressed as a box plot. The upper and lower ends of the Table 6. The quantification limit of RT-qPCR of norovirus box and the horizontal line in the box indicate the first and GI, GII, and GIV.1 were 10 copies/well. The PCR efficiency third quantiles and the median value of data, respectively. of norovirus GI, GII, and GIV.1 were 95.78%, 90.31%, and The lower and higher ends of whiskers indicate the mini- 95.02%, respectively. mum and maximum value of the data, respectively. Isolated data points are outliers. Log reduction of each test material Statistics and Data Analysis for Virucidal Tests was analyzed with the Steel–Dwass non-parametric multiple comparison tests following the Kruskal–Wallis test. Data Numerical values of each well of negative control (PBS) analysis was performed using Microsoft Excel 2010, and and test materials obtained by RT-qPCR were taken for a statistical analysis was performed using the statistical soft- common logarithm. For the numerical values of the test ware SAS 9.2 (SAS Institute Japan) and EXSUS 7.7 soft- materials of the three independent tests, log reduction was ware (CAC Excare Corporation). calculated compared to the negative control, and its average values and SD were calculated. When the numerical value of each well was below the limit of detection (10 copies/ Correlation of Protein Concentration of Stool well in this test), the corresponding well was excluded from Samples and LRVs the subsequent calculation and expressed as BDL (below detection limit). If a BDL value was obtained twice or more This experiment was performed to confirm that fecal materi - in three technical triplicates, the numerical value in the test als did not affect the efficacy of test materials in this study. was BDL. If a BDL value was obtained twice or more in The protein concentrations of stool samples were calcu- three independent tests, the RNA copies number of the test lated by measuring the absorbance at 280 nm by NanoDrop was BDL, and the LRV was set to be greater than the maxi- (Thermo Fisher Scientific, Rockford IL, USA). Correlation mum detectable LRV (ex. > 3.5). In the statistical analysis, between protein concentrations of stool samples and the the numerical processing method was changed. That is, if mean of LRVs for each test material were evaluated with the numerical value of each well was BDL, the value of Spearman’s rank correlation coefficient (correlation coef- the corresponding well was 10. The log reduction of each ficient (r ) and p values). 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Tohoku University and Otsuka Pharmaceutical Factory, Inc., and is PLoS Pathogens, 6(3), e1000831. https ://doi.org/10.1371/journ sponsored by Otsuka Pharmaceutical Factory, Inc. This study may lead al.ppat.10008 31. to the development of products that may be licensed to Otsuka Phar- Bull, R. A., Hansman, G. S., Clancy, L. E., Tanaka, M. M., Rawlinson, maceutical Factory, Inc. After assessment by Tohoku University, this W. D., & White, P. A. (2005). Norovirus recombination in ORF1/ study has been found to have no conflicts of interest. KI, AH and YI ORF2 overlap. Emerging Infectious Diseases, 11(7), 1079–1085. are employees of Otsuka Pharmaceutical Factory, Inc. However, the https ://doi.org/10.3201/eid11 07.04127 3. company had no control over the interpretation of the results or the Campos, C. J. A., & Lees, D. N. (2014). Environmental transmission writing and publication of this study. of human noroviruses in shellfish waters. 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Publisher’s Note Springer Nature remains neutral with regard to https ://doi.org/10.1128/AEM.03802 -14. jurisdictional claims in published maps and institutional affiliations. Sickbert-Bennett, E. E., Weber, D. J., Gergen-Teague, M. F., Sobsey, M. D., Samsa, G. P., & Rutala, W. A. (2005). Comparative efficacy of hand hygiene agents in the reduction of bacteria and viruses. 1 3

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