Inactivation of Human Norovirus Surrogates by Benzalkonium Chloride,Potassium Peroxymonosulfate,Tannic Acid,and Gallic Acid

Abstract

Novel methods to effectively disinfect contact surfaces and prevent human norovirus transmission are essential. The effect of benzalkonium chloride (BAC), potassium peroxymonosulfate (KPMS), tannic acid (TA), and gallic acid (GA) on enteric virus surrogates, murine norovirus (MNV-1), feline calicivirus (FCV-F9), and bacteriophage MS2 was studied. Viruses at high (∼7 log₁₀ PFU/mL) or low (∼5 log₁₀ PFU/mL) titers were mixed with equal volumes of BAC (0.2, 0.5, and 1 mg/mL), KPMS (5, 10, and 20 mg/mL), TA (0.02 and 0.2 mg/mL), GA (0.2, 0.4, and 0.8 mg/mL), or water and incubated for 2 h at room temperature. Viral infectivity after triplicate treatments was evaluated using plaque assays in duplicate. Low titers of FCV-F9 and MNV-1 were completely reduced, while low-titer MS2 was reduced by 1.7–1.8 log₁₀ PFU/mL with BAC at all three concentrations. High-titer FCV-F9 was reduced by 2.87, 3.08, and 3.25 log₁₀ PFU/mL, and high-titer MNV-1 was reduced by 1.55, 2.32, and 2.75 log₁₀ PFU/mL with BAC at 0.1, 0.25, and 0.5 mg/mL, respectively. High-titer MS2 was reduced by ∼2 log₁₀ PFU/mL with BAC at all three concentrations. KPMS at all three concentrations reduced high and low titers of FCV-F9 and MS2 and low-titer MNV-1 to undetectable levels, while high-titer MNV-1 was reduced by 0.92 and 3.44 log₁₀ PFU/mL with KMPS at 2.5 and 5 mg/mL, respectively. TA at 0.2 mg/mL only reduced high-titer FCV-F9 by 0.98 log₁₀ PFU/mL and low-titer FCV-F9 by 1.95 log₁₀ PFU/mL. GA at 0.1, 0.2, and 0.4 mg/mL reduced low-titer FCV-F9 by 2.50, 2.36, and 0.86 log₁₀ PFU/mL, respectively with negligible effects against high-titer FCV-F9. BAC and KPMS show promise to be used as broad-spectrum contact surface disinfectants for prevention of noroviral surrogate contamination.

Introduction

H uman noroviruses are recognized as the leading cause of viral gastroenteritis outbreaks worldwide, with newly emergent strains known to be highly virulent and life threatening to the elderly and immunocompromised (Mead et al., 1999; Siebenga et al., 2009). Transmission of human noroviruses has been shown to occur through person-to person contact, foodborne and waterborne routes, as well as environmental and fomite contamination. Human noroviruses have low infectious doses (10–100 viral particles) and are resistant to environmental degradation and chemical inactivation, making it challenging to find an efficacious method to disinfect contamination associated with human noroviruses (Cheesbrough et al., 2000; Kuusi et al., 2002).

Benzalkonium chloride (BAC) is a quaternary ammonium compound that has wide applications ranging from disinfectant formulations to pharmaceutical preservation (Beasley et al., 1998; Bridier et al., 2011; Marple et al., 2004). As a disinfectant, BAC solutions are readily used in non-alcohol-based hand sanitizers (Moadab et al., 2001; Shintre et al., 2006), in hard surface disinfectants (Carpentier and Cerf, 2011; Gradel et al., 2004), as well as in surgical instrument sterilizing solutions (ASCRS, 2007) and in clinical settings. BAC solutions are found to be effective against bacteria, viruses, and fungi (Bastiani, 1974; Jira et al., 1982; Karabit et al., 1988; Mosca et al., 2006; Thomas et al., 2005). Their antibacterial activity has been reported against Pseudomonas aeruginosa, Streptococcus agalactiae, Staphylococcus aureus, and Escherichia coli among others (Mosca et al., 2006; Richards and Mizrahi, 1978; Thomas et al., 2005). Their antiviral activities have been shown against adenovirus (Lazzaro et al., 2009), acute respiratory syndrome coronavirus (Rabenau et al., 2005), herpes simplex virus (Thompson, 1998), and influenza virus (Armstrong and Froelich, 1964). Interestingly, the effect of BAC against human noroviruses (or its surrogates) has not been well documented in literature.

Potassium peroxymonosulfate (KPMS) compounds have been used as broad-spectrum disinfectants such as in oral hygiene formulations, and pool and spa disinfection (Danner and Merrill, 2006). It is reported that KPMS treatment is effective on both enveloped and non-enveloped viruses (Eleraky et al., 2002). The antiviral effect of KPMS has been reported against feline calicivirus (Eleraky et al., 2002), but the effect of KPMS against MNV-1 and MS2 has not been documented. While BAC and KPMS have been used to decontaminate surfaces against bacteria and some viruses, alternate organic acids are also being researched to compare their effectiveness against pathogens.

Organic acids such as TA and GA are also reported to have antiviral activity against herpes simplex virus, human immunodeficiency virus (HIV-1), human rhinovirus, and hepatitis B virus (Choi et al., 2010; Kratz et al., 2008a,b; Ma et al., 2010; Romero et al., 2005; Xiang et al., 2011). GA has shown anti-fungal, antioxidant, and anti-tumorigenic properties, and antiviral activity (Choi et al., 2010; John and Mukundan, 1979; Konishi and Hotta, 1980; Kratz et al., 2008a, b; Mizuno et al., 1992). TA is associated with antibacterial, antioxidant, and anti-mutagenic properties (Ferguson, 2001; Hadi et al., 2007; Shahidi et al., 1992; Singh et al., 2011). Therefore, the effect of these organic acids against human enteric virus surrogates needs to be determined and compared to existing disinfectants routinely used in industrial and clinical settings.

In the present study, the antiviral effect of BAC at 0.1, 0.25, and 0.5 mg/mL, KPMS at 2.5, 5, and 10 mg/mL, TA at 0.01 and 0.1 mg/mL, and GA at 0.1, 0.2, and 0.4 mg/mL on the infectivity of human norovirus surrogates, feline calicivirus (FCV-F9), and murine norovirus (MNV-1) as well as bacteriophage MS2 was studied.

Methods

Viruses, bacteria, and cell lines

Bacteriophage MS2 and its host E. coli B-15597; FCV-F9, and Crandell Reese Feline Kidney (CRFK) cells were obtained from American Type Culture Collection (ATCC; Manassas, VA). Murine norovirus, MNV-1 was provided as a gift by Dr. Skip Virgin (Washington University, St Louis, MO), and RAW 264.7 cells were obtained from the University of Tennessee at Knoxville.

CRFK and RAW 264.7 cell lines were grown at 37°C with 5% CO₂ in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1× Antibiotic-Antimycotic solution as reported earlier (D'Souza et al., 2006; Su et al., 2010). Viral stocks of FCV-F9 and MNV-1 were prepared by inoculation of FCV-F9 and MNV-1 onto monolayers of CRFK and RAW 264.7 cells, respectively, and incubated until >90% cell lysis. Then the media containing infected cells were freeze-thawed and centrifuged for 10 min at 5,000×g. The supernatants were passed through 0.2-μm membrane filters, aliquoted, and stored at −80°C until use. The viral titer was quantified using plaque assays as described below.

Bacteriophage MS2 was propagated in 6-h E. coli B-15597 host in 3% trypticase soy broth (TSB) containing 0.1% glucose, 2 mM CaCl₂, and 10 μg/mL thiamine (Bae and Schwab, 2008). The bacteriophage was harvested after overnight incubation at 37°C by centrifugation and filtration using the procedure described above for FCV-F9 and MNV-1.

Infectious plaque assays

FCV-F9 and MNV-1 plaque assays were performed in a similar manner as reported earlier (D'Souza et al., 2006; Su et al., 2010):

Step 1: Cells were seeded into six-well plates and incubated until ∼90% confluency.

Step 2: Serial dilutions of treated or untreated viral samples in DMEM-F12 containing FBS was added to wells in duplicate and incubated for 2 h at 37°C in a CO₂ incubator.

Step 3: Inocula were removed, and cells were overlaid with 2 mL of complete DMEM containing 0.75% agarose.

Step 4: Plates were incubated for another 2–3 days and stained with 0.02% neutral red. Plaques were counted after 5-h incubation for MNV-1 and overnight for FCV-F9.

MS2 bacteriophage plaque assay was carried out following the protocol reported earlier (Bae and Schwab, 2008). Briefly, 0.7 mL of serially diluted phage and 0.3 mL of 6-h E. coli B-15597 host were added to 0.6% molten agar, mixed, and poured on tryptic soy agar (TSA) plates. Plates were incubated at 37°C overnight, and plaques were counted.

Antiviral effects of benzalkonium chloride, potassium peroxymonosulfate, gallic acid, and tannic acid

Benzalkonium chloride (MP Biomedicals, LLC, Solon, OH), potassium peroxymonosulfate (TCI America, Portland, OR), gallic acid (ACROS Organics, Fair Lawn, NJ), and tannic acid (Fisher Science Education, Hanover Park, IL) were dissolved in deionized distilled water and sterilized by filtration through 0.22-micron filters. Virus stocks were diluted in phosphate-buffered saline (PBS) to reach titers of ∼7 log₁₀ PFU/mL (high titer) or ∼5 log₁₀ PFU/mL (low titer). Each virus was mixed with an equal volume of BAC at 0.2, 0.5, and 1 mg/mL, KPMS at 5, 10, and 20 mg/mL, TA at 0.02 (pH 5.0) and 0.2 mg/mL (pH 4.6), or GA at 0.2 (pH 3.5), 0.4 (pH 3.3), and 0.8 (pH 3.3) mg/mL, and incubated at room temperature for 2 h. The pH of BAC-virus mixture or TA-virus mixture was found to be neutral. The pH of the tested concentrations of the GA-virus mixture was 6.2–6.7, and the KPMS-virus mixture was pH 4.2–5.0. Individual viruses were mixed with sterile deionized-distilled water and used as the untreated controls. After incubation, treatments were neutralized by 10-fold serial dilution of FCV-F9 and MNV-1 virus in DMEM containing 10% FBS, or MS2 in TSB containing 3% beef extract. All treatments were run in triplicates.

Cytotoxicity assays were run by adding serial dilutions of the chemicals in DMEM-F12 to CRFK and RAW 264.7 cell lines. After 2-h incubation, the cells were overlaid with complete DMEM-F12 containing 0.75% agar and incubated for another 2–3 days. Cytopathic effects were determined by visual inspection under the optical microscope after neutral red staining.

Statistical analysis

Results from triplicate treatments and controls were statistically analyzed using analysis of variance (ANOVA) with SAS software (version 9.2; SAS Institute, Cary, NC) and Tukey's test on a completely randomized design with six sets of data for each treatment condition at α=0.05.

Results and Discussion

In this study, BAC at all three tested concentrations (near the manufacturer's suggested concentrations) was found to be effective in reducing high and low titers of FCV-F9, MNV-1, and MS2 (Table 1). Low titers (5 log₁₀ PFU/mL) of FCV-F9 and MNV-1 were reduced to undetectable levels, while low-titer MS2 was reduced by 1.7–1.8 log₁₀ PFU/mL after 2 h with BAC at all three tested concentrations. BAC at 0.1–0.5 mg/mL was found to be less effective in reducing high-titer compared to low-titer FCV-F9 and MNV-1. High-titer FCV-F9 was reduced by 2.87, 3.08, and 3.25 log₁₀ PFU/mL using BAC at 0.1, 0.25, and 0.5 mg/mL, respectively. High-titer MNV-1 was reduced by 1.55, 2.32, and 2.75 log₁₀ PFU/mL with BAC at 0.1, 0.25, and 0.5 mg/mL, respectively. High-titer MS2 was reduced by ∼2 log₁₀ PFU/mL using BAC at all three concentrations.

Table 1.

Effect of Benzalkonium Chloride (BAC), Potassium Peroxymonosulfate (KPMS), Tannic Acid (TA), and Gallic Acid (GA) on the Infectivity of Feline Calicivirus (FCV-F9), Murine Norovirus (MNV-1), and Bacteriophage MS2 at High Titer (∼7 log ₁₀ PFU/mL) and Low Titer (∼5 log ₁₀ PFU/mL) After 2-h Incubation at Room Temperature

	FCV-F9				MNV-1				MS2
	High titer (log₁₀ PFU/mL)		Low titer (log₁₀ PFU/mL)		High titer (log₁₀ PFU/mL)		Low titer (log₁₀ PFU/mL)		High titer (log₁₀ PFU/mL)		Low titer (log₁₀ PFU/mL)
Treatment	Recovered titer	Reduction	Recovered titer	Reduction	Recovered titer	Reduction	Recovered titer	Reduction	Recovered titer	Reduction	Recovered titer	Reduction
Water	7.11±0.07 A	0	5.03±0.07 A	0	6.59±0.07 A	0	4.79±0.08 A	0	6.87±0.09 A	0	4.80±0.02 A	0
0.1 mg/mL BAC	4.24±0.25 C	2.87	0.00±0.0.00 E	5.03	5.04±0.12 C	1.55	0.00±0.0.00 B	4.79	4.90±0.10 B	1.97	3.06±0.08 B	1.74
0.25 mg/mL BAC	4.03±0.16 CD	3.08	0.00±0.0.00 E	5.03	4.27±0.25 D	2.32	0.00±0.0.00 B	4.79	4.87±0.10 B	2.00	3.03±0.10 B	1.77
0.5 mg/mL BAC	3.86±0.15 D	3.25	0.00±0.0.00 E	5.03	3.84±0.07 E	2.75	0.00±0.00 B	4.79	4.94±0.09 B	1.93	3.06±0.08 B	1.74
2.5 mg/mL KPMS	0.00±0.0.00 E	7.11	0.00±0.0.00 E	5.03	5.67±0.09 B	0.92	0.00±0.0.00 B	4.79	0.00±0.0.00 C	6.87	0.00±0.0.00 C	4.80
5 mg/mL KPMS	0.00±0.0.00 E	7.11	0.00±0.0.00 E	5.03	3.15±0.13 F	3.44	0.00±0.0.00 B	4.79	0.00±0.0.00 C	6.87	0.00±0.0.00 C	4.80
10 mg/mL KPMS	0.00±0.0.00 E	7.11	0.00±0.0.00 E	5.03	0.00±0.00 G	6.59	0.00±0.00 B	4.79	0.00±0.00 C	6.87	0.00±0.00 C	4.80
0.01 mg/mL TA	7.09±0.04 A	0.02	3.16±0.06 C	1.87	—	—	4.77±0.03 A	0.02	—	—	4.81±0.06 A	0.00
0.1 mg/mL TA	6.13±0.09 B	0.98	3.08±0.11 C	1.95	—	—	4.81±0.04 A	0.00±0.00	—	—	4.74±0.10 A	0.06
0.1 mg/mL GA	7.00±0.03 A	0.11	2.53±0.15 D	2.50	—	—	4.88±0.06 A	0.00±0.00	—	—	4.76±0.11 A	0.04
0.2 mg/mL GA	6.98±0.11 A	0.13	2.67±0.16 D	2.36	—	—	4.85±0.05 A	0.00±0.00	—	—	4.78±0.05 A	0.02
0.4 mg/mL GA	7.04±0.03 A	0.07	4.17±0.09 B	0.86	—	—	4.87±0.06 A	0.00±0.00	—	—	4.79±0.11 A	0.01

Within each column, different letters denote significant differences (p<0.05).

KPMS at the three tested concentrations (near the manufacturer's suggested concentrations) reduced FCV-F9 and MS2 at both high and low initial titers to undetectable levels after 2 h at RT (Table 1). The effect of KPMS against high-titer MNV-1 was found to be dose dependent. High-titer MNV-1 was reduced by 0.92, 3.44, and 6.59 log₁₀ PFU/mL after 2-h treatment with 2.5, 5, and 10 mg/mL KMPS at RT, respectively. Low-titer MNV-1 was completely reduced by KMPS at all three tested concentrations.

Treatment with both organic acids (TA and GA) at room temperature for 2 h caused no reduction of low-titer MNV-1 or MS2. As TA and GA at the tested concentration did not show antiviral effects on low-titer MNV-1 and MS2, their effect against high-titer MNV-1 and MS2 were not tested. However, TA at 0.01 and 0.1 mg/mL caused 0.02 (no reduction) and 0.98 log₁₀ PFU/mL reduction of high-titer FCV-F9; and 1.87 and 1.95 log₁₀ PFU/mL reduction of low-titer FCV-F9, respectively (Table 1). GA at 0.1, 0.2, and 0.4 mg/mL caused 2.50, 2.36, and 0.86 log₁₀ PFU/mL reduction of low-titer FCV-F9, respectively, with negligible effects against high-titer FCV-F9. The decreased reduction obtained by GA at the higher concentration of 0.4 mg/mL could potentially be due to aggregation or clumping of the viruses in the presence of 0.4 mg/mL GA. Alternately, some other mechanism could play a role, and this needs further investigation by transmission electron microscopy and by in-depth structural studies.

BAC has been shown to have antiviral effects against both enveloped and non-enveloped viruses (Belec et al., 2000; Wood and Payne, 1998). At room temperature, BAC at 2 mg/mL was shown to reduce enveloped viruses, such as herpes simplex virus (HSV) by 4.5 log, and HIV-1 by 1.9 log after 1 min. Under the same conditions, BAC at 2 mg/mL also reduced the non-enveloped human coxsackie virus by 5.1 log within 1 min, without any reported effect on poliovirus or human adenovirus (Wood and Payne, 1998). At room temperature, 1.25 μg/mL BAC was reported to reduce the titers of enveloped viruses (HSV-2, cytomegalovirus, respiratory syncytial virus) by 3.0–3.2 log; and the titers of non-enveloped viruses (adenovirus, enterovirus, and BK virus) by 1.3–1.7 log after 1 h (Belec et al., 2000). Thus, enveloped viruses were found to be more sensitive to BAC than non-enveloped viruses. The non-enveloped viruses required longer contact times of 1 h to achieve merely 1–1.7 log reduction. Human enteric surrogates FCV-F9, MNV-1, and MS2 are all non-enveloped viruses. Hence, in this study to determine antiviral effects of BAC, KPMS, TA, or GA, longer incubation times of 2 h at room temperature were used to investigate any potential antiviral effects of these four chemicals against human norovirus surrogates.

Even though several mechanisms have been proposed for the antiviral effect of BAC, the actual mechanism of action is not well established. Lazzaro et al. (2009) showed virucidal effects of BAC on adenovirus by immunofluorescence. Wainberg et al. (1990) demonstrated that BAC had inhibitory effects on HIV-1 reverse transcriptase activity. Wood and Payne (1998) found that BAC inactivated HIV-1 and herpes simplex virus viruses by changing the surface components of viruses, making the viruses unable to attach to host cells. Belec et al. (2000) proposed that BAC destroys enveloped virus by ionic detergent effects, which alters viral integrity or potentially virus-host cell interaction for non-enveloped viruses.

KPMS and KPMS containing disinfectants have been shown to have antiviral activities against feline herpes virus, FCV, feline parvovirus, ranavirus, adenovirus types 5 and 6, avian influenza virus, and astrovirus (Bryan et al., 2009; Eleraky et al., 2002; McCormick and Maheshwari, 2004; Schultz-Cherry et al., 2001; Suarez et al., 2003). Eleraky et al. (2002) have shown that KPMS at 10 mg/mL reduced feline herpesvirus, FCV, and feline parvovirus from 5–6 log₁₀ 50% cell-culture infectious dose (CCID₅₀) to undetectable levels after 10 min at room temperature. Similar results with FCV-F9 are obtained in this study, though using longer contact times. Bryan et al. (2009) showed that KPMS at 2 and 5 μg/mL was not effective against ranavirus at room temperature after 60 min. Virkon S, a commercially available disinfectant, when used at 1% (containing 2 mg/mL KPMS) concentration resulted in >8 log reduction of ranavirus and >6 log reduction of adenovirus after 1 min (Bryan et al., 2009; McCormick and Maheshwari, 2004; Schultz-Cherry et al., 2001; Suarez et al., 2003). Our results have shown that KPMS at 2.5 mg/mL reduced high- and low-titer FCV-F9 and MS2, and low-titer MNV-1 to undetectable levels after 2 h at room temperature. However, only 1 log reduction was obtained on high-titer MNV-1, which is not surprising due to the known resistance of MNV-1 to many chemical and processing treatment conditions.

GA and TA have been shown to have antiviral activities against influenza virus, human rhinoviruses, herpes simplex virus type 1 and 2 (HSV-1 and HSV-2), and HIV-1 (Choi et al., 2010; Kratz et al., 2008a,b). In our present study, GA and TA showed antiviral effects only against FCV-F9, but not against MNV-1 and MS2. Though both GA and TA are weak acids, the pH of the acid-virus mixtures were close to neutral after mixing these acids with equal volumes of viruses. The pH of the GA-virus mixture at the tested concentrations was 6.2–6.7, and the pH of the TA-virus mixture at the tested concentrations was pH 7.0. Thus, the antiviral effect of these acids against FCV-F9 cannot be solely attributed to the known reported sensitivity of FCV-F9 to pH (Cannon et al., 2006). The mode of action of GA and TA against FCV is suggested to be by inhibition of virus replication, and/or virus attachment to host cells (Choi et al., 2010; Kratz et al., 2008a,b; Zhang et al., 2012).

Quaternary ammonium chloride, QAC (0.8 mg/mL final concentration) reduced MS2 and FCV by 0.63 and 1.9 log₁₀ PFU/mL after 10 min, and Virkon at 1% (containing 5 mg/mL KPMS) reduced MS2 and FCV from 4–5 log₁₀ PFU/mL to undetectable levels after 10 min (Solomon et al., 2009). In comparison, our results showed that 5 mg/mL KPMS decreased both MS2 and FCV-F9 from ∼5 log₁₀ PFU/mL to undetectable levels after 2 h; and 0.5 mg/mL BAC decreased FCV-F9 to undetectable levels and reduced MS2 by 1.9 log₁₀ PFU/mL after 2 h. Our results using BAC showed better reduction than those reported by Solomon et al. (2009), as a longer contact time of 2 h at RT was used in our study. This correlates with the antiviral effect of QAC, which is known to be time-dependent and slow acting. In comparing BAC and KPMS to our previous study on the antiviral effects of trisodium phosphate (TSP) against human norovirus surrogates, TSP at 5% reduced FCV-F9, MNV-1, and MS2 at both high and low titers to undetectable levels after 1 min at room temperature (D'Souza and Su, 2010). Although TSP has been used in food environments, it is not known to be used in clinical settings.

In conclusion, both BAC and KPMS have significant antiviral effects against human norovirus surrogates. BAC at low concentrations is reported to be non-irritant, non-toxic, and non-corrosive to food-contact surfaces/fomites and thus can be safely used as a disinfectant/decontaminant in the food industry as a broad-spectrum antimicrobial agent. However, BAC requires longer contact time to inactivate non-enveloped viruses. Though KPMS is fast acting, it can be corrosive to equipment and surfaces. On the other hand, the organic acids, GA and TA, are reported to be safe and relatively environmentally-friendly compounds compared to synthetic chemicals. However, they showed only some effect against FCV-F9, without any antiviral effects against MNV-1 or MS2. Hence, they may have only limited application when compared to BAC or KPMS. Therefore, it becomes necessary to state the advantages and disadvantages of these decontaminants, before recommendation for use. BAC needs longer contact times and can be inactivated by anionic and nonionic detergents, and by metal ions, thus BAC should not be combined with other disinfectants for subsequent cleaning. KPMS though fast-acting, is corrosive and may pose a handling risk issue at higher concentrations.

These initial studies provide the basic information and insights into the application of current decontaminants for broad-spectrum use to prevent norovirus outbreaks in clinical and/or industrial settings. Further studies will focus on the antiviral effect of BAC and KPMS against foodborne viruses in the presence of food residues on food-contact surfaces and towards understanding their mechanism of action against these viruses using Transmission Electron Microscopy.

Footnotes

Acknowledgments

The funding provided by the Tennessee Agricultural Experiment Station (UT-TEN-HATCH 00391) to carry out this research is gratefully acknowledged.

Disclosure Statement

No competing financial interests exist.

References

[ASCRS] American Society of Cataract Refractive Surgery; American Society of Ophthalmic Registered Nurses. Recommended practices for cleaning and sterilizing intraocular surgical instruments. J Cataract Refract Surg, 2007; 33:1095–1100.

Armstrong

, Froelich

. Inactivation of viruses by benzalkonium chloride. Appl Microbiol, 1964; 12:132–137.

Bae

, Schwab

. Evaluation of murine norovirus, feline calicivirus, poliovirus, and MS2 as surrogates for human norovirus in a model of viral persistence in surface water and groundwater. Appl Environ Microbiol, 2008; 74:477–484.

Bastiani

. Antibacterial, antifungal and antiprotozoal action of a preparation based on benzalkonium chloride. Farmaco Edizione Pratica, 1974; 29:594–601.

Beasley

, Fishwick

, Miles

, Hendeles

. Preservatives in nebulizer solutions: Risks without benefit. Pharmacotherapy, 1998; 18:130–139.

Belec

, Tevi-Benissan

, Bianchi

, Cotigny

, Beumont-Mauviel

, Si-Mohamed

, Malkin

. In vitro inactivation of Chlamydia trachomatis and of a panel of DNA (HSV-2, CMV, adenovirus, BK virus) and RNA (RSV, enterovirus) viruses by the spermicide benzalkonium chloride. J Antimicrob Chemother, 2000; 46:685–693.

Bridier

, Briandet

, Thomas

, Dubois-Brissonnet

. Comparative biocidal activity of peracetic acid, benzalkonium chloride and ortho-phthalaldehyde on 77 bacterial strains. J Hosp Infect, 2011; 78:208–213.

Bryan

, Baldwin

, Gray

, Miller

. Efficacy of select disinfectants at inactivating Ranavirus. Dis Aquat Organ, 2009; 84:89–94.

Cannon

, Papafragkou

, Park

, Osborne

, Jaykus

, Vinjé

. Surrogates for the study of norovirus stability and inactivation in the environment: A comparison of murine norovirus and feline calicivirus. J Food Prot, 2006; 69:2761–2765.

10.

Carpentier

, Cerf

. Review—Persistence of Listeria monocytogenes in food industry equipment and premises. Int J Food Microbiol, 2011; 145:1–8.

11.

Cheesbrough

, Green

, Gallimore

, Wright

, Brown

DWG

. Widespread environmental contamination with Norwalk-like viruses (NLV) detected in a prolonged hotel outbreak of gastroenteritis. Epidemiol Infect, 2000; 125:93–98.

12.

Choi

, Song

, Bhatt

, Baek

. Anti-human rhinovirus activity of gallic acid possessing antioxidant capacity. Phytother Res, 2010; 24:1292–1296.

13.

Danner

, Merrill

. Disinfectants, disinfection and biosecurity in aquaculture. Aquaculture Biosecurity: Prevention, Control, and Eradication of Aquatic Animal Disease. Scarfe

, Lee

, O'Bryen

. Boston, MA: Blackwell Publishing Professional, 2006; 91–128.

14.

D'Souza

, Sair

, Williams

, Papafragkou

, Jean

, Moore

, Jaykus

. Persistence of caliciviruses on environmental surfaces and their transfer to food. Int J Food Microbiol, 2006; 108:84–91.

15.

D'Souza

, Su

. Efficacy of chemical treatments against murine norovirus, feline calicivirus, and MS2 bacteriophage. Foodborne Pathog Dis, 2010; 7:319–326.

16.

Eleraky

, Potgieter

LND

, Kennedy

. Virucidal efficacy of four new disinfectants. J Am Anim Hosp Assoc, 2002; 38:231–234.

17.

Ferguson

. Role of plant polyphenols in genomic stability. Mutat Res, 2001; 475:89–111.

18.

Gradel

, Sayers

, Davies

. Surface disinfection tests with Salmonella and a putative indicator bacterium, mimicking worst-case scenarios in poultry houses. Poult Sci, 2004; 83:1636–1643.

19.

Hadi

, Bhat

, Azmi

, Hanif

, Shamim

, Ullah

. Oxidative breakage of cellular DNA by plant polyphenols: A putative mechanism for anticancer properties. Semin Cancer Biol, 2007; 17:370–376.

20.

Jira

, Panzig

, Pohloudekfabini

. On the antimicrobial activity of benzalkonium chloride in potential contact lens fluids. Pharmazie, 1982; 37:587–590.

21.

John

, Mukundan

. Virus inhibition by tea, caffeine and tannic acid. Indian J Med Res, 1979; 69:542–545.

22.

Karabit

, Juneskans

, Lundgren

. Studies on the evaluation of preservative efficacy. 3. The determination of antimicrobial characteristics of benzalkonium chloride. Int J Pharm, 1988; 46:141–147.

23.

Konishi

, Hotta

. On the mechanism of inactivation of chikungunya virus by tannic acid. Microbiol Immunol, 1980; 24:847–859.

24.

Kratz

, Andrighetti-Frohner

, Kolling

, Leal

, Cirne-Santos

. Yunes RA, Nunes RJ, Trybala E, Bergstrom T, Frugulhetti I, Barardi CRM, Simoes CMO. Anti-HSV-1 and anti-HIV-1 activity of gallic acid and pentyl gallate. Mem Inst Oswaldo Cruz, 2008a. 103:437–442.

25.

Kratz

, Andrighetti-Frohner

, Leal

, Nunes

, Yunes

, Trybala

, Bergstrom

, Barardi

CRM

, Simoes

CMO

. Evaluation of anti-HSV-2 activity of gallic acid and pentyl gallate. Biol Pharm Bull, 2008b. 31:903–907.

26.

Kuusi

, Nuorti

, Maunula

, Minh

NNT

, Ratia

, Karlsson

, Von Bonsdorff

. A prolonged outbreak of Norwalk-like calicivirus (NLV) gastroenteritis in a rehabilitation centre due to environmental contamination. Epidemiol Infect, 2002; 129:133–138.

27.

Lazzaro

, Abulawi

, Hajee

. In vitro cytotoxic effects of benzalkonium chloride on adenovirus. Eye Contact Lens, 2009; 35:329–332.

28.

, Diao

, Zhao

, Li

, Kang

. A new alternative to treat swine influenza A virus infection: Extracts from Terminalia chebula Retz. Afr J Microbiol Res, 2010; 4:497–499.

29.

Marple

, Roland

, Benninger

. Safety review of benzalkonium chloride used as a preservative in intranasal solutions: An overview of conflicting data and opinions. Otolaryngol Head Neck Surg, 2004; 130:131–141.

30.

McCormick

, Maheshwari

. Inactivation of adenovirus types 5 and 6 by Virkon S. Antiviral Res, 2004; 64:27–33.

31.

Mead

, Slutsker

, Dietz

, McCaig

, Bresee

, Shapiro

, Griffin

, Tauxe

. Food-related illness and death in the United States. Emerg Infect Dis, 1999; 5:607–625.

32.

Mizuno

, Uchino

, Toukairin

, Tanabe

, Nakashima

, Yamamoto

, Ogawara

. Inhibitory effect of tannic acid sulfate and related sulfates on infectivity, cytoeffect, and giant cell formation of human immunodeficiency virus. Planta Med, 1992; 58:535–539.

33.

Moadab

, Rupley

, Wadhams

. Effectiveness of a nonrinse, alcohol-free antiseptic hand wash. J Am Podiatr Med Assoc, 2001; 91:288–293.

34.

Mosca

, Russo

, Miragliotta

. In vitro antimicrobial activity of benzalkonium chloride against clinical isolates of Streptococcus agalactiae. J Antimicrob Chemother, 2006; 57:566–568.

35.

Rabenau

, Kampf

, Cinatl

, Doerr

. Efficacy of various disinfectants against SARS coronavirus. J Hosp Infect, 2005; 61:107–111.

36.

Richards

RME

, Mizrahi

. Differences in antibacterial activity of benzalkonium chloride. J Pharm Sci, 1978; 67:380–383.

37.

Romero

, Efferth

, Serrano

, Castano

, Macias

RIR

, Briz

, Marin

JJG

. Effect of artemisinin/artesunate as inhibitors of hepatitis B virus production in an “in vitro” replicative system. Antiviral Res, 2005; 68:75–83.

38.

Schultz-Cherry

, King

, Koci

. Inactivation of an astrovirus associated with poult enteritis mortality syndrome. Avian Dis, 2001; 45:76–82.

39.

Shahidi

, Wanasundara

, Hong

. Antioxidant activity of phenolic compounds in meat model systems. ACS Symp Ser, 1992; 506:214–222.

40.

Shintre

, Gaonkar

, Modak

. Efficacy of an alcohol-based healthcare hand rub containing synergistic combination of farnesol and benzethonium chloride. Int J Hyg Environ Health, 2006; 209:477–487.

41.

Siebenga

, Vennema

, Zheng

, Vinje

, Lee

, Pang

, Ho

ECM

, Lim

, Choudekar

, Broor

, Halperin

, Rasool

NBG

, Hewitt

, Greening

, Jin

, Duan

, Lucero

, O'Ryan

, Hoehne

, Schreier

, Ratcliff

, White

, Iritani

, Reuter

, Koopmans

. Norovirus illness is a global problem: Emergence and spread of Norovirus GII.4 variants, 2001–2007. J Infect Dis, 2009; 200:802–812.

42.

Singh

, Singh

, Goel

. Traditional uses, phytochemistry and pharmacology of Ficus religiosa: A review. J Ethnopharmacol, 2011; 134:565–583.

43.

Solomon

, Fino

, Wei

, Kniel KE Comparative susceptibilities of hepatitis

A virus

. feline calicivirus, bacteriophage MS2, bacteriophage Phi X-174 to inactivation by quaternary ammonium, oxidative disinfectants. J Antimicrob Agents, 2009; 33:288–289.

44.

, Howell

, D'Souza

. The effect of cranberry juice and cranberry proanthocyanidins on the infectivity of human enteric viral surrogates. Food Microbiol, 2010; 27:535–540.

45.

Suarez

, Spackman

, Senne

, Bulaga

, Welsch

, Froberg

. The effect of various disinfectants on detection of avian influenza virus by real time RT-PCR. Avian Dis, 2003; 47:1091–1095.

46.

Thomas

, Russell

, Maillard

. Antimicrobial activity of chlorhexidine diacetate and benzalkonium chloride against Pseudomonas aeruginosa and its response to biocide residues. J Appl Microbiol, 2005; 98:533–543.

47.

Thompson

. Antiviral activity of Viracea against acyclovir susceptible and acyclovir resistant strains of herpes simplex virus. Antiviral Res, 1998; 39:55–61.

48.

Wainberg

, Spira

, Bleau

, Thomas

. Inactivation of human immunodeficiency virus type-1 in tissue culture fluid and in genital secretions by the spermicide benzalkonium chloride. J Clin Microbiol, 1990; 28:156–158.

49.

Wood

, Payne

. The action of three antiseptics/disinfectants against enveloped and non-enveloped viruses. J Hosp Infect, 1998; 38:283–295.

50.

Xiang

, Mo

, Qu

, Xu

, Jiang

. Ellagic acid from the dried fruits of Canarium album with antihepatitis B activity. Asian J Chem, 2011; 23:3759–3760.

51.

Zhang

, Dai

, Zhong

, Tan

, Lv

, Zhou

, Jiang

. Tannic acid inhibited norovirus binding to HBGA receptors, a study of 50 Chinese medicinal herbs. Bioorg Med Chem, 2012; 20:1616–1323.