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Disinfection efficacy and mechanism of olanexidine gluconate against norovirus

  • Kaoru Imai
    Affiliations
    Naruto Research Institute, Research and Development Center, Otsuka Pharmaceutical Factory, Inc., Naruto, Tokushima, Japan

    Department of Frontier Sciences for Advanced Environment, Graduate School of Environmental Studies, Tohoku University, Sendai, Miyagi, Japan
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  • Makoto Tanaka
    Affiliations
    Naruto Research Institute, Research and Development Center, Otsuka Pharmaceutical Factory, Inc., Naruto, Tokushima, Japan
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  • Seiji Miyoshi
    Affiliations
    Naruto Research Institute, Research and Development Center, Otsuka Pharmaceutical Factory, Inc., Naruto, Tokushima, Japan
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  • Ren Murakami
    Affiliations
    Naruto Research Institute, Research and Development Center, Otsuka Pharmaceutical Factory, Inc., Naruto, Tokushima, Japan
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  • Akifumi Hagi
    Affiliations
    Naruto Research Institute, Research and Development Center, Otsuka Pharmaceutical Factory, Inc., Naruto, Tokushima, Japan
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  • Sachi Yamagawa
    Affiliations
    Naruto Research Institute, Research and Development Center, Otsuka Pharmaceutical Factory, Inc., Naruto, Tokushima, Japan
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  • Daisuke Sano
    Correspondence
    Address correspondence to Daisuke Sano, PhD, Department of Civil and Environmental Engineering, Graduate School of Engineering, Tohoku University, Aoba 6-6-06, Aramaki, Aoba-ku, Sendai, Miyagi 980-8579, Japan.
    Affiliations
    Department of Frontier Sciences for Advanced Environment, Graduate School of Environmental Studies, Tohoku University, Sendai, Miyagi, Japan

    Department of Civil and Environmental Engineering, Graduate School of Engineering, Tohoku University, Sendai, Miyagi, Japan
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Open AccessPublished:December 01, 2021DOI:https://doi.org/10.1016/j.ajic.2021.11.020

      Highlights

      • Antiseptics are important for viral disease prevention and control.
      • Olanexidine gluconate formulation for hand hygiene was effective against selected adenoviruses and calciviruses.
      • Olanexidine gluconate affected the structures of the viruses capsid.
      • The virucidal effect was not affected by organic contaminants.
      • The irritation score of Olanexidine gluconate was within the acceptable level.

      Abstract

      Background

      The purpose of this study was to evaluate the virucidal activity of a new olanexidine-containing formulation for hand hygiene (olanexidine gluconate hand rub; OLG-HR) against non-enveloped viruses and to understand its mechanism of action.

      Methods

      The virucidal activities of OLG-HR against two strains of caliciviruses and three adenovirus serotypes were evaluated through suspension tests. Also, virus-like particles were used to predict the effect of olanexidine gluconate on virus particle structure.

      Results

      The results of suspension tests under conditions with and without interfering substances (1.5% BSA) indicated that OLG-HR had a broad-spectrum effect against non-enveloped viruses, and the virucidal effect was unaffected by organic contaminants. Furthermore, olanexidine inhibited the binding ability of virus-like particles to the binding receptor of human norovirus and increased the aggregation of virus-like particles in a dose-dependent manner. Transmission electron microscopy showed that the morphology of the virus-like particles was affected by exposure to olanexidine, indicating that the protein-denaturing effect of olanexidine gluconate caused the loss of receptor-binding capability of the viral capsid protein.

      Conclusions

      This study suggests that olanexidine gluconate is a potential biological and environmental disinfectant against norovirus and adenovirus.

      Key Words

      Abbreviations:

      OLG (olanexidine gluconate), OLG-HR (olanexidine gluconate hand rub), VLPs (virus-like particles), LRV (log reduction value), FCV (feline calicivirus), MNV (murine norovirus), ADV (adenovirus), BZC-AL (0.2% benzalkonium chloride-containing ethanol), EtOH-A (acidic ethanol containing phosphoric acid), Base (base component of OLG-HR)

      Introduction

      Olanexidine gluconate [OLG; 1-(3,4-dichlorobenzyl)-5-octylbiguanide gluconate] is a novel antibiotic compound developed by Otsuka Pharmaceutical Factory, Inc. An aqueous formulation of 1.5% olanexidine has been used clinically in Japan for preoperative skin preparation [Olanedine Antiseptic Solution 1.5%]. OLG is a biguanide compound and a chlorhexidine digluconate, but its spectrum differs between bacteria and viruses. OLG was more effective than chlorhexidine digluconate and povidone iodine (PVP-I) against methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci both in vitro and in mouse-contaminated skin models.
      • Inoue Y
      • Hagi A
      • Nii T
      • Tsubotani Y
      • Nakata H
      • Iwata K.
      Novel antiseptic compound OPB-2045G shows potent bactericidal activity against methicillin-resistant Staphylococcus aureus and vancomycinresistant Enterococcus both in vitro and in vivo: a pilot study in animals.
      In clinical trials, Obara et al reported that 1.5% OLG significantly reduced the occurrence of overall surgical site infections and superficial surgical site infections compared with PVP-I in clean-contaminated surgery.
      • Obara H
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      Aqueous olanexidine versus aqueous povidone-iodine for surgical skin antisepsis on the incidence of surgical site infections after clean-contaminated surgery: a multicentre, prospective, blinded-endpoint, randomised controlled trial.
      Additionally, the findings of previous studies showed that 1.5% olanexidine-containing 70% ethanol formulation for hand hygiene (OLG hand rub; OLG-HR) was more virucidal than ethanol against human noroviruses.
      • Imai K
      • Hagi A
      • Inoue Y
      • Amarasiri M
      • Sano D.
      Virucidal efficacy of olanexidine gluconate as a hand antiseptic against human norovirus.
      ,
      • Imai K
      • Hagi A
      • Yamagawa S
      • Sano D.
      Olanexidine gluconate formulations as environmental disinfectants for enveloped viruses infection control.
      The importance of antiseptics in infection control, particularly emerging viral infections, cannot be overemphasized. Although it is necessary to use approved antiseptics to prevent disease transmission, the virucidal efficacy of commonly available antiseptics on viruses is not always well understood. In a previous study using culture-independent methods, we reported that OLG was effective against norovirus; however, culture-dependent methods have not been used to examine the virucidal efficacy of OLG against non-enveloped viruses, other than murine norovirus in the absence of interfering substance.
      • Imai K
      • Hagi A
      • Inoue Y
      • Amarasiri M
      • Sano D.
      Virucidal efficacy of olanexidine gluconate as a hand antiseptic against human norovirus.
      The structural units of non-enveloped viruses include nucleic acids (genomic DNA or RNA) and capsid proteins that protect the viral genome. Disinfectants inactivate viruses through several mechanisms, including through the destruction of the viral genome and/or the outer structure of viruses.
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      UV irradiation leads to nucleic acid destruction,
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      whereas iodine seems to inactivate viruses by impairing their ability to attach to host cells.
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      Non-enveloped viruses such as norovirus and adenovirus (ADV) are considered to be relatively highly resistant to antiseptics because of the robust structure of the outer capsid proteins, which play a pivotal role in the binding of non-enveloped viruses to host cells. Some ADV serotypes attack host animals by binding to the coxsackievirus-ADV receptor of the host cells through the activities of capsid fiber proteins.
      • Roelvink PW
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      • et al.
      The coxsackievirus-adenovirus receptor protein can function as a cellular attachment protein for adenovirus serotypes from subgroups A, C, D, E, and F.
      Regarding human noroviruses, volunteer studies have shown that secretor-positive individuals are more susceptible to human norovirus.
      • Indesmith LISAL
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      Studies using intestinal organoids have shown that cells expressing histo-blood group antigens can be efficiently bound to virus-like particles (VLPs), which are genetically engineered virions.
      • Rimkute I
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      Histo-blood group antigens of glycosphingolipids predict susceptibility of human intestinal enteroids to norovirus infection.
      VLPs do not replicate in infected cells, because they do not have a genome, but are structurally similar to infectious virions. VLPs are used for vaccine development and drug delivery systems.
      • Deo VK
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      Additionally, human norovirus-like particles are useful in studying the efficacy of disinfectants against human noroviruses that do not have a simple culture and growth procedure.
      • Sato J
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      Effects of disinfectants against norovirus virus-like particles predict norovirus inactivation.
      Previous reports have shown that OLG affects bacterial and viral proteins.
      • Imai K
      • Hagi A
      • Inoue Y
      • Amarasiri M
      • Sano D.
      Virucidal efficacy of olanexidine gluconate as a hand antiseptic against human norovirus.
      ,
      • Hagi A
      • Iwata K
      • Nii T
      • Nakata H
      • Tsubotani Y
      • Inoue Y.
      Bactericidal effects and mechanism of action of olanexidine gluconate, a new antiseptic.
      So, we estimated the dose-dependent effects of olanexidine gluconate on VLPs from the viewpoints of the binding ability to HBGA, cohesiveness, and structures.
      In this study, the virucidal efficacy of OLG-HR against non-enveloped viruses was evaluated using a culture-dependent method with two strains of human norovirus surrogate viruses and three serotypes of ADVs. To test the effect of OLG on the capsid structure of non-enveloped virus, norovirus-like particles were exposed to OLG, and the denaturing effect was observed by transmission electron microscopy. Based on these results, we discussed the modes of action of olanexidine gluconate on non-enveloped viruses.

      Material and methods

      Animal experiments were conducted with permission from the Animal Experiment Committee in accordance with the Guidelines on Animal Experiments of the Naruto Research Institute.

      Test viruses and cells

      Feline calicivirus ATCC VR-782 (FCV), ADV type 2 ATCC VR-846 (ADV2), type 5 ATCC VR-5 (ADV5), and type 7 ATCC VR-7 (ADV7) were purchased from American type culture collection (ATCC; VA). Murine norovirus S99 (MNV) was purchased from Friedrich-Loeffler-Institut (FLI; Greifswald Island Riems, Germany). CRFK (National Institutes of Biomedical Innovation, Health and Nutrition JCRB Cell Bank, Osaka, Japan), RAW264.7 (Public Health England, Salisbury, UK), and A549 (JCRB Cell Bank, Osaka, Japan) cells were used as hosts for FCV, MNV, and the three strains of ADV, respectively. Host cells were grown in Dulbecco's modified Eagle's medium (DMEM; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) containing 10% fetal bovine serum (Invitrogen, CA). Viral stocks were inoculated into each host for 1 hour and then cultured for 3 days after changing the medium. Three freeze-thaw cycles were performed, and the culture was centrifuged (9680 × g, 4°C, 1 hour). The supernatants of MNV and FCV were then collected and ultracentrifuged at 111,000 × g for 90 minutes at 4°C in a Beckman 50.2 Ti rotor (Beckman Coulter, Inc., CA). The pellets were resuspended in PBS and passed through a filter with a pore size of 0.45 μm (Millipore, MA). The solutions were stored at -70°C and used as test virus solutions for the suspension test (without 1.5% BSA). Test solutions for FCV or MNV and 30% BSA solution (Sigma-Aldrich, MO) were mixed in equal volumes and used as test virus solutions in suspension tests under conditions with 1.5% BSA. The culture supernatants of the three serotypes of ADV were collected and concentrated approximately five times by ultrafiltration (Amicon Ultra - 15 10 K centrifugal filter devices; Merck KGaA, Germany). The concentrated solutions were stored at -70°C for further analysis.

      Experimental animal

      Female SPF white rabbits (Kbl:JW, KITAYAMA LABES Co., Ltd., Nagano, Japan) were used in this study. All animals were housed in a pathogen-free environment in the laboratory at the Naruto Research Institute in Otsuka Pharmaceutical Factory, Inc., and received sterile food and water.

      Suspension test

      A suspension test was conducted under conditions with and without interfering substances (1.5% BSA) with some modifications to the various standard test methods described previously.

      European Committee for Standardization. EN14476: Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of virucidal activity in the medical area -Test method and requirements (Phase 2/Step 1) 2013.

      • Blümel J
      • Glebe D
      • Neumann-Haefelin D
      • et al.
      Guideline of “Deutsche Vereinigung zurBeka¨mpfung der Viruskrankheiten e.V.” (DVV; German Association for the Control of Virus Diseases) and Robert Koch-Institute(RKI; German Federal Health Authority) for testing the virucidalefficacy of chemical disinfectants in the human medical area.

      American Society for Testing and Materials. ASTM E1052-11: Standard test method to assess the activity of microbicides against viruses in suspension. 2011.

      The test substances were OLG-HR, 0.2% benzalkonium chloride-containing ethanol (BZC-AL, Maruishi Pharmaceutical Co. Ltd, Osaka, Japan), acidic ethanol containing phosphoric acid (EtOH-A, Saraya Co., Ltd., Osaka, Japan), and base component of OLG-HR (Base). Briefly, 20 µL of test virus solution was mixed with 180 μL of test material (or PBS as a negative control) followed by incubation for 15, 30, and 60 seconds at room temperature (setting at 23°C). After the reactions, the test substances were inactivated by the neutralization method (for FCV and ADV) or the gel filtration method (for MNV). For the neutralization method, 100 μL of the reaction mixtures were mixed with 900 μL of neutralizer solution (Composition: 1.67 w/v% K2HPO4, 0.06 w/v% KH2PO4, 0.1 v/v% Triton X-100, 1.17 w/v% Soy lecithin, 10 w/v% polysorbate 80, 0.5 w/v% sodium thiosulfate hydrate, and 1 w/v% Tamol NN8906; pH adjusted to 7.8-7.9). In the gel filtration method, 100 μL of the reaction mixture was mixed with 900 μL of PBS and immediately sieved through a gel filtration column (PD-10 Empty Columns filled with Sephacryl S-400; GE Healthcare, NJ).
      • Imai K
      • Hagi A
      • Inoue Y
      • Amarasiri M
      • Sano D.
      Virucidal efficacy of olanexidine gluconate as a hand antiseptic against human norovirus.
      ,

      European Committee for Standardization. EN14476: Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of virucidal activity in the medical area -Test method and requirements (Phase 2/Step 1) 2013.

      ,
      • Eterpi M
      • McDonnell G
      • Thomas V.
      Virucidal activity of disinfectants against parvoviruses and reference viruses.
      The neutralized solutions were serially diluted 10-fold with culture media six times. Suspension tests were performed using three biological replicates at each time point.
      In the cytotoxicity test, the cell viability against the negative control (PBS) at the lowest dilution ratio of each test substance excluding the virus was evaluated using WST-8 assay (Cell Counting Kit-8, Dojindo Laboratories, Kumamoto, Japan). In the neutralizing test, before mixing the test substance and virus, the test substance with the neutralizer or gel filtration column was inactivated, and the viral titer was compared with that of the negative control.

      Plaque assay

      Plaque assay was conducted as previously reported.
      • Gonzalez-Hernandez MB
      • Bragazzi Cunha J
      • Wobus CE
      Plaque assay for murine norovirus.
      Host cells on six-well plates were inoculated with 500 μL of 10-fold dilution series of each reaction solution and incubated at room temperature for 1 hours (three technical replicates). After removing the reaction solution, an agar medium was added, and the plate was then incubated for 2 d (FCV and MNV) or 10 d (ADV) at 37°C. Subsequently, 2 mL 0.01% neutral red solution (Sigma-Aldrich, St Louis, MO) was added, followed by incubation for 4 hours at 37°C. Thereafter, the neutral red solution was removed, and the plaques were visually counted. Viral titers (log10 PFU/mL) were measured and log reduction values (LRVs) were calculated after all virucidal tests following EN14476,

      European Committee for Standardization. EN14476: Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of virucidal activity in the medical area -Test method and requirements (Phase 2/Step 1) 2013.

      and results were expressed as the mean of the log10 PFU/mL ± 95% confidence interval (CI).

      VLP production and purification

      VLP genotypes GII.3 (AB758450), GII.4 (AB668028), and GII.6 (AB758451) were prepared according to published method.
      • Miura T
      • Sano D
      • Suenaga A
      • et al.
      Histo-blood group antigen-like substances of human enteric bacteria as specific adsorbents for human noroviruses.
      • Kitamoto N
      • Tanaka T
      • Natori K
      • et al.
      Cross-reactivity among several recombinant calicivirus virus-like particles (VLPs) with monoclonal antibodies obtained from mice immunized orally with one type of VLP.
      • Pogan R
      • Schneider C
      • Reimer R
      • Hansman G
      • Uetrecht C.
      Norovirus-like VP1 particles exhibit isolate dependent stability profiles.
      These genotypes were selected because they are the most prevalent GII noroviruses. Recombinant capsid proteins were expressed in Sf9 cells, after which VLPs secreted into the cell medium were collected by centrifugation at 9,600 × g for 60 minutes. VLPs were concentrated by ultracentrifugation at 100,000 g in an SW28 rotor (Beckman, Palo Alto, CA). VLPs were purified by CsCl ultracentrifugation. Purified VLPs were resuspended in PBS (pH 6.5) and quantified using the Lowry method. The concentration was adjusted to 1 mg/mL using PBS (pH 6.5).

      Suspension test of VLPs

      Briefly, 20 µL of test VLPs was mixed with 180 μL of test substances (or PBS as a negative control), followed by incubation for 30 s at room temperature. After incubation, 200 μL of the reaction solution was mixed with 1800 μL of PBS (pH 6.5). The entire reaction solution was added to the dialysis membrane (Spectra/Por RC, MWCO: 3,500-5,000, Spectrum Laboratories, HOU) and dialyzed with 100 mL PBS (pH 6.5) for 2 hours. The dialysate was exchanged and dialyzed for another 2 hours. Dialysis liquid was used for subsequent evaluation.

      Porcine gastric mucin (PGM)-binding assay

      PGM-binding assay was performed as previously described.
      • Lindesmith LC
      • Costantini V
      • Swanstrom J
      • et al.
      Emergence of a norovirus GII.4 strain correlates with changes in evolving blockade epitopes.
      • Lindesmith LC
      • Beltramello M
      • Donaldson EF
      • et al.
      Immunogenetic mechanisms driving norovirus GII.4 antigenic variation.
      • Lindesmith LC
      • Ferris MT
      • Mullan CW
      • et al.
      Broad blockade antibody responses in human volunteers after immunization with a multivalent norovirus VLP candidate vaccine: immunological analyses from a phase I clinical trial.
      Briefly, 100 μL of porcine mucin (mucin from porcine stomach, type III, Sigma-Aldrich, MO) suspended in 10 µg/mL of PBS was added to a 96-well clear plate (Corning incorporated, NY) and reacted at room temperature for 4 hour. The plate was washed three times with PBS containing 0.01% Tween 20 (PBST), followed by the addition of 100 µL of 5% BSA/PBST and stored overnight at 4°C (PGM plate). After dialysis, the reaction solution was added to the PGM plate (n = 3) and stored for 1 hour at 37°C. After washing three times with PBST, anti-VLP antiserum (rabbit polyclonal antibody, made in-house) was added as the first antibody, and the plate was incubated for 2 hours at 37°C. After washing three times with PBST, horseradish peroxidase-conjugated goat anti-rabbit IgG (Abcam, Cambridge, MA) was added as a secondary antibody, followed by incubation for 1 hour at room temperature. After washing three times with PBST, TMB solution (SeraCare Life Sciences, Inc., MA) was added, followed by incubation for 10 minutes at room temperature. Thereafter, stop solution (SeraCare Life Sciences, Inc., MA) was added, and the absorbance at 450 nm (measurement wavelength) and 600 nm (reference wavelength) was determined using a microplate reader (ARVO, PerkinElmer, Inc., MA). The value of the reference wavelength was subtracted from the value of the measurement wavelength, and the relative values to negative control were presented as the mean ± standard deviation (SD).

      Dynamic light scattering

      VLP aggregate size was determined using dynamic light scattering (DLS). Briefly, 1 µL of the dialysis liquid was added to the cuvette, and the particle size was measured at room temperature using ELS-6100 (Otsuka Electronics Co., Ltd., Osaka, Japan). The ELS software calculated the size distribution of particles in each sample from the light scattering intensity data. The median diameters of the values obtained in the three independent tests were calculated and presented as mean ± SD.
      Thereafter, the relationship between the DLS and the PGM-binding assay data was determined using Spearman's correlation.

      Transmission electron microscopy (TEM)

      The dialysis liquid was adsorbed onto coated copper grids (Nisshin EM CO., Ltd., Tokyo, Japan) for 15 minute and stained with 2% phosphotungstic solution (pH 7.0) for 15 seconds. Excess solution was removed with filter paper, and the TEM image was acquired using a transmission electron microscope HT7800 (Hitachi High-Tech Corporation, Tokyo, Japan.) at 120 kV.

      Rabbit skin cumulative irritation test

      For the skin irritation test, 50 µL of OLG-HR or BZC-AL was applied to each administration site (2 cm × 2 cm) on the rabbit skin (n = 3) after hair clipping. After contact for 24 hours, the test substance was washed with cotton balls soaked in warm water. This operation was performed for 14 days consecutively. The administration start date was set as day 0. The administration sites were observed before the removal of test substances. The scores were calculated according to the criteria shown in Table 2, and the mean value was calculated.
      • Draize JH
      • Woodard G
      • Herbert C.
      Methods for the study of irritation and toxicity of substances applied topically to the skin and mucous membranes.

      Data analysis

      Data collected during the study were analyzed using Microsoft Excel 2013, SAS (version 9.4; SAS Institute Japan), and EXSUS 10.0.3 software (CAC Croit Corporation). In PGM-binding assay and DLS, the mean values of each group were compared with PBS groups using Dunnett's test, and significance was set at P < .05, P < .01, and P < .001, indicated as *, **, and ***, respectively.

      Result and discussion

      Virucidal efficacy of OLG against norovirus surrogate viruses and ADVs

      In this study, the virucidal efficacy of OLG-HR against two norovirus surrogate viruses and three ADV serotypes was evaluated using suspension tests. Although there is no evidence of a relationship between the viral titers obtained under experimental conditions and viral infection prevention in real life scenarios, some guidelines state that LRV ≥4 is an index of the virucidal efficacy of the drug or test material in the suspension test.

      European Committee for Standardization. EN14476: Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of virucidal activity in the medical area -Test method and requirements (Phase 2/Step 1) 2013.

      ,
      • Blümel J
      • Glebe D
      • Neumann-Haefelin D
      • et al.
      Guideline of “Deutsche Vereinigung zurBeka¨mpfung der Viruskrankheiten e.V.” (DVV; German Association for the Control of Virus Diseases) and Robert Koch-Institute(RKI; German Federal Health Authority) for testing the virucidalefficacy of chemical disinfectants in the human medical area.
      The cytotoxicity tests showed that the difference in cell viability of test substance-exposed groups compared with that of the negative control was within ±25%, and the neutralizing tests showed that the differences in the infectious viral titer from the initial titer were within 0.5 log10 TCID50/mL (Tables S1 and S2). This indicated that the virucidal activity of the test substances could be evaluated through the virucidal efficacy experiment.
      The results of the suspension tests are shown in Table 1. Under conditions without 1.5% BSA, the LRVs of OLG-HR against the two human norovirus surrogate viruses and three ADV serotypes were >4 at the three time points examined. In contrast, the LRVs of BZC-AL and EtOH-A varied depending on the virus type. The LRV of BZC-AL against MNV was >4 at the three time points examined, whereas the LRV of BZC-AL against FCV increased with exposure duration, with the lowest and highest values (3.71 ± 0.48 and 5.11 ± 0.29, respectively; LRV ± SD) obtained at 15 and 60 seconds of exposure, respectively. The results of the virucidal efficacy tests of OLG-HR against MNV under clean condition (without 1.5% BSA) were same with our previous report.
      • Imai K
      • Hagi A
      • Inoue Y
      • Amarasiri M
      • Sano D.
      Virucidal efficacy of olanexidine gluconate as a hand antiseptic against human norovirus.
      Regarding the three ADV serotypes, the LRV of BZC-AL against ADV2 was <4 at the three time points examined, whereas the LRV of BZC-AL against ADV5 was >4 at 30 and 60 seconds of exposure. Additionally, the LRV of BZC-AL against ADV7 was <4 at 15 and 30 seconds of exposure. The disinfectant resistance of adenoviruses was serotype-dependent, but OLG-HR showed a relatively strong efficacy against all 3 serotypes.
      Table 1Evaluation of virucidal efficacy of antiseptics against non-enveloped viruses using suspension test
      Testsubstance1.5%BSAMean of log reduction values± 95% CI, n = 3
      Feline calicivirus F9Murine norovirus S99Adenovirus type 2Adenovirus type 5Adenovirus type 7
      15 s30 s60 s15 s30 s60 s15 s30 s60 s15 s30 s60 s15 s30 s60 s
      OLG-HR-5.22
      Upper detection limit.
      ± 0.08
      5.22
      Upper detection limit.
      ± 0.08
      5.22
      Upper detection limit.
      ± 0.08
      4.86
      Upper detection limit.
      ± 0.02
      4.86
      Upper detection limit.
      ± 0.02
      4.86
      Upper detection limit.
      ± 0.02
      4.79 ± 0.516.50 ± 0.686.73 ± 0.216.29
      Upper detection limit.
      ± 0.24
      6.29
      Upper detection limit.
      ± 0.24
      6.29
      Upper detection limit.
      ± 0.24
      4.91 ± 0.975.14 ± 0.725.46 ± 0.44
      +2.64 ± 0.224.90
      Upper detection limit.
      ± 0.14
      4.90
      Upper detection limit.
      ± 0.14
      4.48
      Upper detection limit.
      ± 0.15
      4.48
      Upper detection limit.
      ± 0.15
      4.48
      Upper detection limit.
      ± 0.15
      ---------
      BZC-AL-3.71 ± 0.484.00 ± 1.935.11 ± 0.294.86
      Upper detection limit.
      ± 0.02
      4.86
      Upper detection limit.
      ± 0.02
      4.86
      Upper detection limit.
      ± 0.02
      0.38 ± 0.221.48 ± 0.603.04 ± 0.873.17 ± 0.906.29
      Upper detection limit.
      ± 0.24
      6.29
      Upper detection limit.
      ± 0.24
      1.00 ± 0.502.53 ± 1.374.14 ± 0.57
      +1.11 ± 0.182.07 ± 1.163.97 ± 1.424.48
      Upper detection limit.
      ± 0.15
      4.48
      Upper detection limit.
      ± 0.15
      4.48
      Upper detection limit.
      ± 0.15
      ---------
      EtOH-A-------1.44 ± 0.556.16 ± 1.616.73 ± 0.214.59 ± 1.696.29
      Upper detection limit.
      ± 0.24
      6.29
      Upper detection limit.
      ± 0.24
      5.56
      Upper detection limit.
      ± 0.34
      5.56
      Upper detection limit.
      ± 0.34
      5.56
      Upper detection limit.
      ± 0.34
      Upper detection limit.
      The interference substances (1.5% BSA) were used in the tests using OLG-HR and BZC-AL. The LRV of OLG-HR against FCV showed an increasing trend with increase in exposure duration, with LRVs >4 obtained at 30 and 60 s of exposure. Additionally, the LRV of OLG-HR against MNV was >4 at the three time points examined. Furthermore, the LRV of BZC-AL against FCV was <4 at the three time points examined, whereas the LRV of BZC-AL against MNV was >4 at the three time points examined. Comparing the disinfectant resistance of two norovirus surrogate viruses, FCV tended to be more resistant under contaminating conditions than MNV. Overall, OLG-HR was more effective than the other test materials against the viruses under both clean and contaminated conditions.

      VLP–HBGA binding inhibition by OLG

      HBGA is considered to be one of the cellular receptors for human norovirus; thus, loss of HBGA-binding ability is thought to lead to the loss of virus infectivity. Therefore, the binding ability of human norovirus VLPs to HBGA under antiseptic exposure were examined in this study. Some reports have suggested that the structure of VLPs changes reversibly depending on the external liquid environment.
      • Pogan R
      • Dülfer J
      • Uetrecht C.
      Norovirus assembly and stability.
      • Mertens BS
      • Velev OD.
      Characterization and control of surfactant-mediated norovirus interactions.
      • Shoemaker GK
      • van Duijn E
      • Crawford SE
      • et al.
      Norwalk virus assembly and stability monitored by mass spectrometry.
      In this study, the reversible effect of antiseptics against the VLP structure was inhibited by incubating the VLP suspension in PBS for more than 4 hours after exposure to the test substances. Therefore, the effects of VLPs detected in this study were considered irreversible. First, we evaluated the effect of antiseptics on the binding ability of VLPs of GII.3, GII.4, and GII.6 to HBGA (Fig 1A, 1B, and 1C). OLG-HR, 1.5% OLG, base components of OLG-HR (Base), EtOH, and EtOH-A were used as test substances and PBS was used as a negative control. EtOH, and EtOH-A. EtOH and EtOH-A were selected because they are commonly used as hand sanitizers. The exposure time was fixed at 30 seconds, and the results showed that the antiseptics significantly affected the binding ability of the VLPs to HBGA. Particularly, the two 1.5% OLG formulations exhibited stronger binding inhibitory effect than the other agents against all three genotypes. A comparison of the sensitivities of the three VLP genotypes showed that GII.3 (Fig 1A) was the most sensitive, while GII.6 (Fig 1C) was the least sensitive. Although the sensitivities of the genotypes to the antiseptics were different, the structures of proteins of the three viral genotypes were most affected by OLG-HR.
      Fig 1
      Fig. 1Binding ability of VLPs to HBGA after exposing test substances to VLPs. The change in VLP–HBGA binding ability due to disinfectant exposure for 30 s was detected using PGM-binding assay. A, B, and C show GII.3, GII.4, and GII.6 respectively. The VLP solution after exposure to test substances for 30 s was added to the porcine mucin-layered 96-well plate. After each reaction of the first and secondary antibody, a TMB reaction was performed, and the absorbance at 450 nm (measurement wavelength) and 600 nm (reference wavelength) was measured. The data are presented as mean ± standard deviation (SD; n = 3). The mean values were compared using Dunnett's test, and significance was set at P < .05, P < .01, and P < .001, indicated as *, **, and ***, respectively.

      OLG concentration-dependent inhibition of VLP–HBGA binding and agglutination

      We also investigated the effect of OLG on VLP–HBGA binding and agglutination in a dose-dependent manner against norovirus VLP GII3, GII4, and GII6 (Fig 2A, 2B, and 2C). The most basic formulation of OLG, 1.5% OLG, was used to prepare the dilution series. To achieve this, a 5-fold dilution series of OLG from 1500 µg/mL (1.5% w/v) to 24 µg/mL was prepared using distilled water. In Figure 2, the bar shows the VLP–HBGA binding capacity, and the line shows the size of the VLP measured by DLS. OLG concentration-dependent inhibition of VLP–HBGA binding was observed in the three genotypes. The inhibitory effect of the antiseptic on VLP–HBGA binding was significantly different between the genotypes. Additionally, the VLP particle size was larger with an increase in OLG concentration; however, at 1500 µg/mL higher OLG concentrations, the aggregates were too large to be detected. Also, the effects of OLG on VLP–HBGA agglutination was slightly different among the genotypes. Furthermore, the results of Spearman's correlation analysis showed that there was a strong negative correlation between VLP–HBGA binding and agglutination in the three genotypes. The correlation coefficients were -0.942 (P = .000), -.800 (P = .000), and -0.816 (P = .000), for GII.3, GII.4, and GII.6, respectively.
      Fig 2
      Fig. 2Correlation between particle size and binding ability of VLPs exposed to various doses of olanexidine gluconate. The effects of different OLG concentrations on the HBGA-binding ability of VLPs and the particle size of VLPs were determined using PGM-binding assay and DLS, respectively. A, B, and C show GII.3, GII.4, and GII.6 respectively. The correlation between the HBGA-binding ability of the VLPs and VLP particle size was analyzed. Mean ± SD, n = 3. #Precipitates were observed, and particle size could not be determined by DLS. The mean values were compared using Dunnett's test, and significance was set at P < .05, P < .01, and P < .001, indicated as *, **, and ***, respectively. The correlation between the values of DLS values PGM-binding assay was analyzed using Spearman's correlation coefficient.

      OLG concentration-dependent effect on VLP structure

      Figure 3 shows the morphology of Gll.6 VLP after exposure to different doses of OLG. At relatively low concentrations, such as 24 (B) and 120 µg/mL (C), the shape of Gll.6 VLP appeared partially intact, but as the dose increased, the structure was altered, and aggregates were formed. Although there have been several reports on the effects of disinfectants on VLP structure,
      • Sato J
      • Miki M
      • Kubota H
      • et al.
      Effects of disinfectants against norovirus virus-like particles predict norovirus inactivation.
      ,
      • Broglie JJ
      • Alston B
      • Yang C
      • et al.
      Antiviral activity of gold/copper sulfide core/shell nanoparticles against human norovirus virus-like particles.
      increase in aggregate formation caused by exposure to OLG have not been previously reported. To summarize these results, Figures 1 and 2 indicate that OLG inhibits VLP-HBGA binding activity in a concentration-dependent manner. In addition, Figure 3 shows that the inhibition is caused by the agglutination characteristic and consequent viral particles destruction.
      Fig 3
      Fig. 3Effect of olanexidine gluconate on VLP structure. A typical image of OLG dose-dependent effect on VLP GII.6 structure. A, B, C, D, and E show negative control (PBS), 24, 120, 600, and 3000 µg/mL, respectively. TEM images were obtained by negative staining. Bar = 200 nm.

      Evaluation of skin cumulative irritation caused by OLG

      Rabbit skin irritation experiment was conducted, and the score was calculated according to the criteria listed in Table 2, using the Draize method.
      • Draize JH
      • Woodard G
      • Herbert C.
      Methods for the study of irritation and toxicity of substances applied topically to the skin and mucous membranes.
      The mean values of three independent scores were calculated, but the SD values were not calculated because these values were categorized data. According to Draize's criteria, compounds with mean value ≤2 are mild irritants, those with indices ranging from 2 to 5 are moderate irritants, and those with values >6 are considered severe irritants. These criteria were defined for the determination of primary irritation, but in ISO10993-10, similar criteria were used to classify cumulative irritation.
      International Organization for Standardization
      ISO 10993-10. Biological Evaluation of Medical Devices - Part 10: Tests for Irritation and Skin Sensitization.
      Therefore, the criteria in Table 2 that are based on Draize's and ISO10993-10 indices were used in this study (cumulative irritation score). The average irritant score for each observation day was calculated and is shown in Figure 4, and individual data are shown in Table S3. OLG-HR caused slight erythema on the rabbit's skin at day 1; however, the average score at days 7 and 8 was 2, after which the effect remained the same, with scores <2 after 14 days of treatment. Moreover, no edema was observed at any stage. Additionally, the irritation caused by BZC-AL at the beginning of the application (days 1-3) was lesser than that caused by OLG-HR, but the score increased steadily thereafter, causing edema, which did not heal during the observation period. Draiz's test on rabbit skin can be used to predict the severe irritation potential of a compound on human skin; however, it is difficult to differentiate between mild and moderate irritations.
      • Phillips L
      • Steinberg M
      • Maibach HI
      • Akers WA.
      A comparison of rabbit and human skin response to certain irritants.
      For instance, BZC-AL is considered safe for use as a hand disinfectant. Although the results of the Draiz's test performed in this study cannot be fully extrapolated to human skin, it is considered that the risk of skin irritation by OLG-HR is not high.
      Table 2Evaluation criteria of skin reactions for rabbit skin cumulative irritation test
      A. Erythema and Eschar Formation
        No erythema0
        Very slight erythema (barely perceptible)1
        Well-defined erythema2
        Moderate to severe erythema3
        Severe erythema (beet redness) to slight eschar formation  (injuries in depth)4
         Total possible erythema score4
      B. Edema Formation
        No edema0
        Very slight edema (barely perceptible)1
        Slight edema (edges of area well defined by definite raising)2
        Moderate edema (area raised approximately 1 mm)3
        Severe edema (raised more than 1 mm, and extending beyond area of   exposure)4
         Total possible edema score4
          Total possible score for irritation8
      Fig 4
      Fig. 4Rabbit skin cumulative irritation by OLG-HR. OLG-HR or BZC-AL (50 μL) was applied to the administration area (2 × 2 cm) of three rabbit skins after hair clipping and left for approximately 24 h. After evaluating the irritation scores for each test substance according to the Draize criteria, the test substances were washed off and reapplied (day 1). The same procedure was repeated 14 times. The average values of three independent scores were calculated, but the standard deviations were not calculated because these values were categorized data. A polygonal line with a round symbol indicates the average scores of OLG-HR, and the triangle symbol indicates the average scores of BZC-AL (n = 3).
      Antiseptics for viral infection control should exhibit high efficacy and immediate effect for successful inactivation of viruses. Particularly, since surfaces for disinfection usually contain contaminants, it is necessary to confirm the efficacy of antiseptics under conditions that mimic the actual usage scenario. The results of the present study showed that OLG-HR was highly effective against norovirus surrogate viruses and ADV serotypes in both conditions with and without 1.5% BSA. Combined with the results of our previous study on human noroviruses,
      • Imai K
      • Hagi A
      • Inoue Y
      • Amarasiri M
      • Sano D.
      Virucidal efficacy of olanexidine gluconate as a hand antiseptic against human norovirus.
      OLG-HR is expected to have a strong virucidal efficacy against human norovirus and ADV. However, these results are based on in vitro experiments and may not fully represent real antiseptic usage scenarios. The effectiveness of antiseptics in virus infection control is ultimately demonstrated by the clinical infection prevention effect. Therefore, future studies should examine the viral infection control effect of OLG-HR through clinical trials. Furthermore, the adoption of antiseptics for use against viral infections is not only dependent on its effectiveness but also safety. The findings of the present study showed that although OLG-HR has strong viral protein-denaturing and -aggregating effects, its effect on rabbit skin was mild.
      Understanding the mechanism of action of antimicrobial compounds helps predict their effectiveness as antiseptics and define their usage. It is known that the structure and cohesiveness of virus particles change reversibly depending on the salt and/or pH in the external environment.
      • Gerba CP
      • Betancourt WQ.
      Viral aggregation: impact on virus behavior in the environment.
      Moreover, the structure of norovirus particles is unstable under basic conditions (pH >8).
      • Pogan R
      • Dülfer J
      • Uetrecht C.
      Norovirus assembly and stability.
      ,
      • Mertens BS
      • Velev OD.
      Characterization and control of surfactant-mediated norovirus interactions.
      Additionally, studies have shown that ADV2 and ADV5 can easily diffuse under acidic conditions and aggregate under basic conditions.
      • Kahler AM
      • Cromeans TL
      • Metcalfe MG
      • Humphrey CD
      • Hill VR.
      Aggregation of adenovirus 2 in source water and impacts on disinfection by chlorine.
      ,
      • Rexroad J
      • Evans RK
      • Middaugh CR.
      Effect of pH and ionic strength on the physical stability of adenovirus type 5.
      Furthermore, it has been reported that aggregated virus particles are less sensitive to disinfectants than dispersed particles.
      • Gerba CP
      • Betancourt WQ.
      Viral aggregation: impact on virus behavior in the environment.
      ,
      • Mattle MJ
      • Crouzy B
      • Brennecke M
      • R. Wigginton K
      • Perona P
      • Kohn T
      Impact of virus aggregation on inactivation by peracetic acid and implications for other disinfectants.
      Considering these findings, the olanexidine-concentration-dependent changes in the structure and cohesiveness of viral particles were enhanced by the formulation conditions of OLG-HR (pH = 9.5). This hypothesis is supported by the results of the present study, which showed that OLG-HR has a stronger virucidal activity and a greater inhibitory effect on HBGA than EtOH-A, EtOH, OLG, and Base.

      Conclusion

      This study demonstrates that OLG-HR has strong virucidal efficacy against norovirus and adenovirus. The findings of the study showed that OLG significantly reduced the binding ability of the virus capsid to the binding receptor due to protein denaturation and agglutination. Additionally, the formulation conditions of OLG-HR improved the efficacy of OLG.

      Acknowledgments

      We are deeply grateful to Ms. Eri Yoshikawa of Otsuka Pharmaceutical Factory, Inc. , for English proofreading. We would like to thank Editage (www.editage.com) for English language editing.

      Appendix. SUPPLEMENTARY MATERIALS

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