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Antiviral and Virucidal Activities of N šœ¶ -Cocoyl-L-Arginine Ethyl Ester

Antiviral and Virucidal Activities of N šœ¶ -Cocoyl-L-Arginine... Hindawi Publishing Corporation Advances in Virology Volume 2011, Article ID 572868, 6 pages doi:10.1155/2011/572868 Research Article Antiviral and Virucidal Activities of Nα-Cocoyl-L-Arginine Ethyl Ester 1 1 1, 2 2 Hisashi Yamasaki, Kazuko Tsujimoto, Keiko Ikeda, Yukiko Suzuki, 3 1 Tsutomu Arakawa, and A. Hajime Koyama Division of Virology, Department of Cellular and Molecular Medicine, Graduate School of Medicine, Wakayama Medical University, 580 Mikazura, Wakayama 641-0011, Japan School of Health and Nursing Science, Wakayama Medical University, 580 Mikazura, Wakayama 641-0011, Japan Alliance Protein Laboratories, Thousand Oaks, CA 91360, USA Correspondence should be addressed to A. Hajime Koyama, koyama@wakayama-med.ac.jp Received 20 July 2011; Accepted 5 September 2011 Academic Editor: Jay C. Brown Copyright © 2011 Hisashi Yamasaki et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Various amino acid-derived compounds, for example, Nα-Cocoyl-L-arginine ethyl ester (CAE), alkyloxyhydroxylpropylarginine, arginine cocoate, and cocoyl glycine potassium salt (Amilite), were examined for their virucidal activities against herpes simplex virus type 1 and 2 (HSV-1 and HSV-2), influenza A virus (IAV), and poliovirus type 1 (PV-1) in comparison to benzalkonium chloride (BKC) and sodium dodecylsulfate (SDS) as a cationic and anionic control detergent and also to other commercially available disinfectants. While these amino acid-derived compounds were all effective against HSV-1 and HSV-2, CAE and Amilite were the most effective. These two compounds were, however, not as effective against IAV, another enveloped virus, as against HSV. Cytotoxicity of CAE was weak; at 0.012%, only 5% of the cells were killed under the conditions, in which 100% cells were killed by either SDS or BKC. In addition to these direct virucidal effects, CAE inhibited the virus growth in the HSV-1- or PV-1-infected cells even at 0.01%. These results suggest a potential application of CAE as a therapeutic or preventive medicine against HSV superficial infection at body surface. 1. Introduction most of these disinfectants are generally toxic to cells and tissues, when come in contact with skins and in particular Recently there has been an increasing necessity for a multi- mucosal surfaces, limiting their in vivo applications. We tude of preparedness against infectious diseases. Every year have observed that aqueous arginine solution inactivates we encounter emerging and reemerging infectious diseas- various enveloped viruses, although relatively high arginine es. While vaccination is a primary strategy to overcome concentration was required [5–10]. Such enhanced virus viral diseases [1], vaccines are not readily available for inactivation by arginine was considered applicable for clear- many viruses and, even if available, not accessible for every ance of contaminated viruses in biopharmaceutical products. individual due to various reasons (e.g., cost, distribution, Antiviral activity of arginine opened a window for topical and politics). Another compounding problem with virus applications against superficial infectious diseases where infection is that antiviral drugs are not available for many both virucidal and antiviral activities might reduce the viral viral diseases [2]. Disinfection at body surface, such as hand load in the infected mucosal cells. Here we have undertaken sanitization, has been an effective measure for the prevention a systematic screening of amino acid derived compounds in against viral diseases and even applicable for the therapy of search for effective, nontoxic antiviral and virucidal agents, certain viral diseases, provided that its tissue toxicity can be which can be used for both hand sanitization and superficial tolerated [3]. infections on body surface. Several available compounds A number of compounds, including organic solvents and were tested for their effects on inactivation of herpes simplex detergents, are known to inactivate viruses [4]. However, viruses, influenza virus, and poliovirus and their toxic effects 2 Advances in Virology on cultured cells and compared with the commercially 2.4. Effectofthe Reagentonthe Multiplication of theVirus. available known disinfectants. Monolayered cells in 35 mm dishes were infected with the virus at an indicated multiplicity of infection (MOI). The infected cells were further incubated at 37 C (for HSV-1, 2. Materials and Methods HSV-2, and IAV) or 35.5 C (for PV-1) for the indicated period in the serum-free MEM (pH 7.4) containing 0.1% 2.1. Cells and Viruses. MDCK, HEp-2, and Vero cells were BSA and the indicated concentrations of the test compounds. grown in Eagle’s minimum essential medium (MEM) con- The compounds were added at the onset of the incubation. taining 5% fetal bovine serum. Herpes simplex virus type 1, For the experiments with influenza virus, acetylated trypsin strain F, (HSV-1), herpes simplex virus type 2, strain 186, (4 μg/mL) was also added to the medium for the proteolytic (HSV-2), influenza virus A/Aichi/68 (IAV) and poliovirus activation of virus infectivity. At the indicated time, the type 1 (PV-1), Sabin vaccine strain, were used throughout the culture medium was harvested and the amount of total experiments [11–13]. The viruses were propagated in Vero progeny virus in the culture was determined as described cells (for HSV-1, HSV-2, and PV-1) in MEM supplemented previously [11–13]. with 0.5% fetal bovine serum (FBS) or in MDCK cells (for IAV) in MEM supplemented with 0.1% bovine serum albumin (BSA) and acetylated trypsin (4 μg/mL). The viruses 2.5. Assay for the Cytotoxicity. Confluent monolayers of Vero were stored at −80 C until use. The amount of each virus was cells were incubated in 0.85% sodium chloride solution (pH measured by a plaque assay as described previously [11–13]. 5.5) containing 20 mM MES (2-Morpholinoethanesulfonic acid) and varying concentrations of each test compound at room temperature for 15 min. To determine the extents 2.2. Reagents. L-arginine hydrochloride (referred as of cell death, monolayered cells were trypsinized to obtain arginine), butyroyl-L-arginine (ButArg), Nα-cocoyl- single cell suspension. After the addition of MEM containing L-arginine ethyl ester (CAE), N-[3-alkyl(12,14)oxy-2- 10% calf serum to the suspension to neutralize the trypsin hydroxypropyl]-L-arginine hydrochloride (Amisafe LMA- and to stabilize the cells, the number of the living or dead 60), L-arginine cocoate (Aminosoap AR-12), potassium cells was determined by a dye exclusion method with trypan Nα-Cocoyl-L-glycinate (Amilite GCK-12K), benzalkonium blue as described previously [15]. chloride (BKC), sodium dodecylsulfate (SDS), sodium Nα-dodecanoylsarcosinate (LS), Chlorhexidine gluconate (CHX), ethylalchol (EtOH), povidone iodine (PI), and 3. Results and Discussion sodium hypochlorite (NaOCl) were of biochemical research grade and kindly provided by Ajinomoto Co., Inc (Tokyo, 3.1. Effect of Amino Acid Derived Compounds on the Infectivity Japan). The compounds with trademark are used as ingre- of DNAand RNAViruses. We examined the virucidal dients for cosmetic products. The pH of each solvent was activity of arginine and amino acid derived compounds (i.e., adjusted to the desired values using pH electrode type 6378 0.5 M arginine, 0.5 M ButArg, 0.2% CAE, 0.2% Amisafe, and pH meter type F-54 (Horiba; Kyoto, Japan). A pH meter 0.2% Aminosoap, and 0.2% Amilite) on viruses of three was manually calibrated every 2 h during the preparation of completely different kinds; that is, HSV-1 and HSV-2 (Family test solutions. of Herpesviridae), IAV (Family of Orthomyxoviridae), and PV-1 (Family of Picornaviridae). Both HSVs and IAV are 2.3. Assay for Virucidal Activity. All the starting materials large enveloped viruses and need the cell nucleus for the were stored on ice prior to the virus inactivation exper- virus multiplication: the former has a double-stranded DNA iments. An excess volume of solutions was mixed with genome, and the latter has a segmented negative-stranded the virus stock so that both the pH and concentration of RNAgenome[16, 17]. In contrast to the viruses of these two the test solutions would not be affected: that is, a 190 μL virus families, PV-1 is a small nonenveloped virus carrying a positive-stranded RNA as a genome and replicates in the aliquot of the solutions received a 10 μL aliquot of virus 7 8 preparations (approximately 10 ∼10 plaque-forming units cytoplasm of the infected cells [18]. [PFU]/mL). This virus preparation was incubated at the Figure 1 shows the effects of these compounds on the indicated temperature for 5 min. After incubation, the test infectivity of HSV-1, when the virus was incubated at 30 C mixtures were chilled in ice-water bath, and, immediately, for 5 min. As a control, the virus was incubated in PBS aliquots of these virus samples were 100-fold diluted with and the relative residual infectivity for each compound was ice-cold Dulbecco’s phosphate-buffered saline (PBS) without calculated by dividing the number of the infectious virus in 2+ 2+ Ca and Mg containing 0.1% BSA for IAV or 0.5% the test solutions by that of the PBS control. As shown in FBS for other viruses to stop the virus inactivation. The Figure 1, HSV-1 was sensitive to these compounds except viruses were further diluted to yield virus counts suitable for Amisafe; the virus infectivity decreased below or near −5 for measurements, and the number of infectious virus in the detection level (10 of the control). In the case of the treated preparation was measured by a plaque assay on Amisafe, the decrease in the virus infectivity was marginally Vero (for HSV-1, HSV-2, and PV-1) or MDCK (for IAV) less than that incubated in the PBS. These results clearly show cells. There was little virus inactivation in PBS, and hence that arginine and amino acid derived compounds (except the amount of infectious virus in PBS was used as a control Amisafe) can efficiently inactivate HSV-1, an enveloped DNA count [14]. virus, under the experimental conditions. Although pH, ion Advances in Virology 3 0.1 0.1 0.01 0.01 0.001 0.001 0.0001 0.0001 0.00001 0.00001 Figure 1: Inactivation of HSV-1. The virus was incubated in the Figure 2: Inactivation of IAV. See the legend to Figure 1.Black and solution indicated below for 5 min at 30 C. Number of infectious white bars represent the results in the presence or absence of 0.1% viruswas determinedbyaplaqueassayonVerocells afterthe BSA. incubation and was normalized to that incubated in PBS. Arginine (0.5 M at pH 4.0 in 10 mM citrate), ButArg (0.5 M in pH 4.0 in 10 mM citrate), CAE (0.2% in 5 mM citrate and 0.15 M NaCl at of 0.1% BSA resulted in suppression, at least partially, of pH 4.8), Amisafe (0.2% in 5 mM citrate and 0.15 M NaCl at pH IAV inactivation by CAE, Amisafe, Amilite, BKC, SDS, and 4.8), Aminosoap (0.2% in 5 mM citrate at pH 7.0), Amilite (0.2% LS (black columns in Figure 2). This is unlikely due to the in 5 mM citrate at pH 8.0), BKC (0.1% in 5 mM citrate at pH 7.0), interaction of BSA with the virus, as it did not affect the virus SDS (0.2% in 5 mM citrate at pH 7.0), LS (0.2% in 5 mM citrate infectivity in PBS. It is most likely due to the ability of BSA to at pH 7.0), CHX (0.5% at pH 6.0), EtOH (70% in 5 mM citrate at bind fatty acids [19] and hence these compounds that have pH 7.0), PI (0.23% in 5 mM citrate at pH 3.5), NaOCl (100 ppm in 5 mM phosphate at pH 7.0), and PBS (pH 7.4). nonpolar moieties. The observed effects of these compounds on IAV suggest the potential mechanism of inactivation. Comparison of species, or ionic strength of the solution can affect the degree arginine, ButArg, CAE, Aminosoap, BKC, EtOH, PI, and of virus inactivation [14], we have shown before that HSV- NaOCl, which showed strong virucidal effects on this 1 is relatively stable above pH 4.8 and the effects of pH 4.0 virus, demonstrates no distinct pattern with compound alone are much weaker than the combined effects of the pH structure. Arginine and ButArg are known to suppress and arginine or ButArg [5, 6]. The results were compared protein-protein interactions, probably through their ability with commercial disinfectants (i.e., 0.1% BKC, 0.2% SDS, to interact with proteins [20–22]. CAE, Aminosoap, and 0.2% LS, 0.5% CHX, 70% EtOH, 0.23% PI, and 100 ppm BKC have surfactant properties. EtOH is an organic solvent. NaOCl). These commercial disinfectants also showed strong These compounds can interact with not only proteins, but virus inactivation on HSV-1 (Figure 1), although the effect also lipid membranes. PI and NaOCl are strong oxidants of PI may be entirely due to the low pH (i.e., pH 3.5). and can interact with most components of virus particles. Enveloped viruses are known to be sensitive to detergents, On the contrary, there appears to be a unique structure which can perturb and solubilize lipid bilayers in the viral pattern for less active compounds, that is, Amisafe, Amilite, envelope. In agreement with this, HSV-2, another human SDS, LS, and CHX. These are all detergents, meaning that herpesvirus of different serotype and pathogenicity, also detergent-like properties are insufficient for the effective showed a similar sensitivity profile to these amino acid virus inactivation. However, these detergents are effective derivatives and disinfectants (data not shown). against HSV-1, demonstrating that the virucidal effectiveness Next, we tested these compounds in Figure 1 against is virus dependent. In contrast to these enveloped viruses, IAV, an enveloped virus of another virus family. As shown poliovirus was resistant to inactivation by these amino in Figure 2 (white columns), the profile was quite different acid derived compounds, as only negligible degree of the from that observed for HSV-1 (and HSV-2). ButArg, CAE, inactivation was observed (Figure 3). Even combination of Aminosoap, BKC, EtOH, PI, and NaOCl reduced the IAV pH 4.0 and arginine or ButArg was insufficient. The lack −5 infectivity below the detection level (10 of the control). of sensitivity of PV-1 may suggest that inactivation of Arginine, Amisafe, Amilite, SDS, LS, and CHX showed a HSV-1 (Figure 1)and IAV(Figure 2) by these compounds varying degree of virucidal activities, with Amisafe being requires the presence of lipid envelope on the structure significantly less effective, similar to its effect on HSV-1, and of target viruses. It should be noteworthy that although with CHX being least effective (white columns in Figure 2). these compounds appear to act on lipids, it does not We have shown before that the pH profile of IAV inactivation necessarily mean solubilization of envelope lipid bilayer. The is complex [14] and, at least, a part of inactivation by low concentrations of the detergents used in these experiments pH samples (arginine, ButArg, and PI) is due to the pH, (0.2%) are below critical micelle concentrations (CMCs). but that the pH alone cannot explain the strong inactivation Although the CMCs of ionic detergents are generally affected by arginine and ButArg [5, 6]. Interestingly, the addition by the pH of detergent solutions, they are considered to Residual infectivity Arginine ButArg. CAE Amisafe Aminosoap Amilite BKC SDS LS CHX EtOH PI NaOCl PBS Residual infectivity Arginine ButArg. CAE Amisafe Aminosoap Amilite BKC SDS LS CHX EtOH PI NaOCl PBS 4 Advances in Virology 1 10 0.1 0.01 0.1 0.001 0.0001 0.01 0.00001 0.001 Figure 3: Inactivation of poliovirus. See the legend to Figure 1. 0.0001 0.00001 be in the range of ∼10 mM (approximately 0.4%) under 0 0.01 0.02 0.03 0.04 the experimental conditions. Below CMC, detergents are CAE (%) unlikely capable of destroying membrane structure. Figure 4: Inactivation of viruses by CAE. The viruses were Among these compounds, CAE and Aminosoap showed incubated at 20 C for 5 min in 10 mM citrate buffer (pH 5.0) a marked virucidal activity. Considering that CAE has been containing 0.15 M sodium chloride and varying concentrations of reported to have bacteriocidal activity [23], we further CAE. Number of infectious virus was determined by a plaque characterized the virucidal activity of CAE. assay after the incubation and was normalized to that incubated in PBS (pH 7.4). Circles ()for HSV-1; blackcircle(•)for HSV-2; 3.2. Characterization of Virucidal Activity of CAE. Figure 4 triangles () for IAV; squares ()for PV-1. shows the effects of CAE concentration on the inactivation of HSV-1, HSV-2, IAV, and PV-1. As CAE was more soluble required higher concentrations to achieve a similar level of at acidic pH, we used pH 5.0 in these experiments. This inactivation under these conditions. pH alone caused little inactivation for HSVs (less than 20% inactivation, if any) and PV-1 (almost no inactivation), but a 3.3. Cytotoxic Effects of the Reagents. The above three com- significant inactivation for IAV (50 ∼ 70% inactivation) [14]. pounds were then compared for cell toxicity. As shown in This is probably due to acid-induced conformational change Figure 6, BKC showed a strong cytocidal effect. When the of HA spike proteins present in IAV envelope [17]. Under monolayers of Vero cells were incubated in isotonic buffer the concentration range (0.001 to 0.04% CAE), PV-1 showed solution in the presence of various concentrations of CAE, only a marginal loss of the infectivity, while IAV showed a BKC, and SDS for 15 min at room temperature and numbers small, but significant (60% decrease at 0.04% CAE), decrease of dead and alive cells were determined by dye exclusion in infectivity with increasing CAE concentrations. In contrast test with trypan blue, BKC showed a drastic increase in the to these two viruses, both HSV-1 and HSV-2 showed a number of dead cells at the concentration above 0.002%. At sharp decrease in infectivity, leading to 10 -fold reduction the concentration required for the effective virucidal effect at 0.02% CAE for HSV-1 and ∼10 -fold reduction at 0.01% (0.003%), BKC killed more than 10% of the cells. In contrast CAE for HSV-2. Although both HSV-1 and HSV-2 belong to BKC, cell death by SDS and CAE occurred at 2.5- and to the same virus family, HSV-2 showed a significantly 10-fold higher concentrations, indicating their lower cell stronger inactivation than HSV-1 at low CAE concentrations. toxicity. It should be noted that SDS solubilized plasma membrane of the cells at the cytotoxic concentrations while This virucidal activity of CAE was slightly, but significantly, CAE or BKC did not (data not shown), leading to a possible enhanced by lowering the pH of the test solutions to 4.0 or 4.5; for example, the inactivation of HSV-1 or HSV-2 at underestimation of the fraction of dead cells in the SDS- treated dishes. The results suggest that at 0.01% CAE, about 0.005% CAE was only marginal at pH 5.0, but was enhanced 2% of the cells were killed by the compound (Figure 6), while 10-fold (for HSV-1) or 100-fold (for HSV-2) at pH 4.0 at the same CAE concentration. more than 99% of the virus was inactivated (Figure 5). To further characterize the virucidal effects of CAE, it was compared with SDS, an anionic detergent, and BKC, 3.4. Inhibition of Virus Multiplication by CAE. The results in a cationic detergent, at neutral pH. Figure 5 shows such Figures 1–5 show in vitro virus inactivation. Whether the a comparison of virucidal activities against HSV-2. Both same compounds can yield a reduced virus multiplication BKCand SDSshowedmarkedeffects (more than two-log in in vivo experiments was then examined by treating the reduction) on HSV-2 infectivity even at low concentrations, infected cells with them. Figure 7 shows the results of CAE for example, at 0.002% and 0.003%, respectively, while CAE and BKC on the relative virus yields of HSV-1 (circle) or Residual infectivity Arginine ButArg. CAE Amisafe Aminosoap Amilite BKC SDS PBS Relative virus yield Advances in Virology 5 0.1 0.1 0.01 0.01 0.001 0.0001 0 0.005 0.01 0.001 Reagent (%) Figure 5: Effects of CAE, BKC, and SDS on the infectivity of HSV-2. HSV-2 was incubated in PBS (pH 7.4) containing varying 0.0001 concentrations of each compound at 30 C for 5 min. The amounts 0 0.005 0.01 of infectious progeny viruses were determined by a plaque assay Reagent (%) after the incubation and were normalized to that incubated in PBS (pH 7.4). Circles () for CAE; triangles ()for BKC; squares() for SDS. Figure 7: Effect of CAE and BKC on the virus multiplication. Confluent monolayers of HEp-2 cells were infected with HSV-1 (, •) at an MOI of 14 or PV-1 (, ) at an MOI of 9. The infected cells were incubated for overnight in the medium containing varying concentrations of CAE (, )orBKC (•, )at37 Cfor HSV-1or at 35.5 C for PV-1. At the end of infection (20 h after infection for HSV-1 and 16 h after infection for PV-1), the amounts of infectious 0.1 progeny viruses were determined and were normalized to the virus yield in the absence of these compounds. 0.01 inhibit the multiplication of two viruses widely different in the way the replication and transcription of the viral genome occur (i.e., in the nucleus or in the cytoplasm of the infected 0.001 cells). It is interesting to note that BKC and CAE, which were 0 0.01 0.02 both ineffective even at 0.1 and 0.2% in inactivating PV-1, Reagent (%) can suppress the growth of the same virus in the infected Figure 6: Effect of CAE, BKC, and SDS on the cell viability. cells. It is evident that the decreased virus yield in infected Confluent monolayers of Vero cells were incubated in 0.85% cells for PV-1 (and perhaps HSV-1) is not simply due to the sodium chloride solution (pH 5.5) containing 20 mM MES and virus inactivation. varying concentrations of each compound at room temperature Generally, virus-inactivating agents (disinfectants) show for 15 min. The treated cells were trypsinized to obtain a single toxicity to cells and tissues, limiting their applications. cell suspension and the amounts of the live and dead cells in each We have undertaken a systematic screening of amino acid culture were determined by a dye exclusion test with trypan blue. derived compounds in search for effective, nontoxic virus- Circles () for CAE; triangles ()for BKC; squares() for SDS. inactivating agents and found that, against HSV-1 and HSV- 2, CAE has notable activities to inactivate the infectivity PV-1 (triangle), when the virus-infected cells were incubated of extracellular virus particles and to suppress the virus in the medium containing varying concentration of CAE multiplication in the infected cells at the concentration with (open symbol) or BKC (closed symbol): note that the highest tolerable cytotoxic effect. These results may support a future concentrations used are still below the concentrations used potential application of CAE as a therapeutic or preventive for virus inactivation. The multiplication of these two viruses medicine against HSV superficial infection at body surface, was much more strongly suppressed by BKC than CAE. such as herpetic keratitis and genital herpes. However, their effects on these two viruses were different. Namely, CAE suppressed more strongly the virus yield of PV-1 than HSV-1, opposite to BKC. Comparison of the Conflict of Interests results in Figure 6 suggests that the suppression of these virus multiplications by CEA or BKC was not due to the death of The authors do not have commercial or other associations the infected cells. These results show that both BKC and CEA that might pose a conflict of interests. Residual infectivity Fraction of dead cells Relative virus yields 6 Advances in Virology Acknowledgments [16] B. Roizman and D. M. 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Antiviral and Virucidal Activities of N šœ¶ -Cocoyl-L-Arginine Ethyl Ester

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Copyright Ā© 2011 Hisashi Yamasaki et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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10.1155/2011/572868
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Hindawi Publishing Corporation Advances in Virology Volume 2011, Article ID 572868, 6 pages doi:10.1155/2011/572868 Research Article Antiviral and Virucidal Activities of Nα-Cocoyl-L-Arginine Ethyl Ester 1 1 1, 2 2 Hisashi Yamasaki, Kazuko Tsujimoto, Keiko Ikeda, Yukiko Suzuki, 3 1 Tsutomu Arakawa, and A. Hajime Koyama Division of Virology, Department of Cellular and Molecular Medicine, Graduate School of Medicine, Wakayama Medical University, 580 Mikazura, Wakayama 641-0011, Japan School of Health and Nursing Science, Wakayama Medical University, 580 Mikazura, Wakayama 641-0011, Japan Alliance Protein Laboratories, Thousand Oaks, CA 91360, USA Correspondence should be addressed to A. Hajime Koyama, koyama@wakayama-med.ac.jp Received 20 July 2011; Accepted 5 September 2011 Academic Editor: Jay C. Brown Copyright © 2011 Hisashi Yamasaki et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Various amino acid-derived compounds, for example, Nα-Cocoyl-L-arginine ethyl ester (CAE), alkyloxyhydroxylpropylarginine, arginine cocoate, and cocoyl glycine potassium salt (Amilite), were examined for their virucidal activities against herpes simplex virus type 1 and 2 (HSV-1 and HSV-2), influenza A virus (IAV), and poliovirus type 1 (PV-1) in comparison to benzalkonium chloride (BKC) and sodium dodecylsulfate (SDS) as a cationic and anionic control detergent and also to other commercially available disinfectants. While these amino acid-derived compounds were all effective against HSV-1 and HSV-2, CAE and Amilite were the most effective. These two compounds were, however, not as effective against IAV, another enveloped virus, as against HSV. Cytotoxicity of CAE was weak; at 0.012%, only 5% of the cells were killed under the conditions, in which 100% cells were killed by either SDS or BKC. In addition to these direct virucidal effects, CAE inhibited the virus growth in the HSV-1- or PV-1-infected cells even at 0.01%. These results suggest a potential application of CAE as a therapeutic or preventive medicine against HSV superficial infection at body surface. 1. Introduction most of these disinfectants are generally toxic to cells and tissues, when come in contact with skins and in particular Recently there has been an increasing necessity for a multi- mucosal surfaces, limiting their in vivo applications. We tude of preparedness against infectious diseases. Every year have observed that aqueous arginine solution inactivates we encounter emerging and reemerging infectious diseas- various enveloped viruses, although relatively high arginine es. While vaccination is a primary strategy to overcome concentration was required [5–10]. Such enhanced virus viral diseases [1], vaccines are not readily available for inactivation by arginine was considered applicable for clear- many viruses and, even if available, not accessible for every ance of contaminated viruses in biopharmaceutical products. individual due to various reasons (e.g., cost, distribution, Antiviral activity of arginine opened a window for topical and politics). Another compounding problem with virus applications against superficial infectious diseases where infection is that antiviral drugs are not available for many both virucidal and antiviral activities might reduce the viral viral diseases [2]. Disinfection at body surface, such as hand load in the infected mucosal cells. Here we have undertaken sanitization, has been an effective measure for the prevention a systematic screening of amino acid derived compounds in against viral diseases and even applicable for the therapy of search for effective, nontoxic antiviral and virucidal agents, certain viral diseases, provided that its tissue toxicity can be which can be used for both hand sanitization and superficial tolerated [3]. infections on body surface. Several available compounds A number of compounds, including organic solvents and were tested for their effects on inactivation of herpes simplex detergents, are known to inactivate viruses [4]. However, viruses, influenza virus, and poliovirus and their toxic effects 2 Advances in Virology on cultured cells and compared with the commercially 2.4. Effectofthe Reagentonthe Multiplication of theVirus. available known disinfectants. Monolayered cells in 35 mm dishes were infected with the virus at an indicated multiplicity of infection (MOI). The infected cells were further incubated at 37 C (for HSV-1, 2. Materials and Methods HSV-2, and IAV) or 35.5 C (for PV-1) for the indicated period in the serum-free MEM (pH 7.4) containing 0.1% 2.1. Cells and Viruses. MDCK, HEp-2, and Vero cells were BSA and the indicated concentrations of the test compounds. grown in Eagle’s minimum essential medium (MEM) con- The compounds were added at the onset of the incubation. taining 5% fetal bovine serum. Herpes simplex virus type 1, For the experiments with influenza virus, acetylated trypsin strain F, (HSV-1), herpes simplex virus type 2, strain 186, (4 μg/mL) was also added to the medium for the proteolytic (HSV-2), influenza virus A/Aichi/68 (IAV) and poliovirus activation of virus infectivity. At the indicated time, the type 1 (PV-1), Sabin vaccine strain, were used throughout the culture medium was harvested and the amount of total experiments [11–13]. The viruses were propagated in Vero progeny virus in the culture was determined as described cells (for HSV-1, HSV-2, and PV-1) in MEM supplemented previously [11–13]. with 0.5% fetal bovine serum (FBS) or in MDCK cells (for IAV) in MEM supplemented with 0.1% bovine serum albumin (BSA) and acetylated trypsin (4 μg/mL). The viruses 2.5. Assay for the Cytotoxicity. Confluent monolayers of Vero were stored at −80 C until use. The amount of each virus was cells were incubated in 0.85% sodium chloride solution (pH measured by a plaque assay as described previously [11–13]. 5.5) containing 20 mM MES (2-Morpholinoethanesulfonic acid) and varying concentrations of each test compound at room temperature for 15 min. To determine the extents 2.2. Reagents. L-arginine hydrochloride (referred as of cell death, monolayered cells were trypsinized to obtain arginine), butyroyl-L-arginine (ButArg), Nα-cocoyl- single cell suspension. After the addition of MEM containing L-arginine ethyl ester (CAE), N-[3-alkyl(12,14)oxy-2- 10% calf serum to the suspension to neutralize the trypsin hydroxypropyl]-L-arginine hydrochloride (Amisafe LMA- and to stabilize the cells, the number of the living or dead 60), L-arginine cocoate (Aminosoap AR-12), potassium cells was determined by a dye exclusion method with trypan Nα-Cocoyl-L-glycinate (Amilite GCK-12K), benzalkonium blue as described previously [15]. chloride (BKC), sodium dodecylsulfate (SDS), sodium Nα-dodecanoylsarcosinate (LS), Chlorhexidine gluconate (CHX), ethylalchol (EtOH), povidone iodine (PI), and 3. Results and Discussion sodium hypochlorite (NaOCl) were of biochemical research grade and kindly provided by Ajinomoto Co., Inc (Tokyo, 3.1. Effect of Amino Acid Derived Compounds on the Infectivity Japan). The compounds with trademark are used as ingre- of DNAand RNAViruses. We examined the virucidal dients for cosmetic products. The pH of each solvent was activity of arginine and amino acid derived compounds (i.e., adjusted to the desired values using pH electrode type 6378 0.5 M arginine, 0.5 M ButArg, 0.2% CAE, 0.2% Amisafe, and pH meter type F-54 (Horiba; Kyoto, Japan). A pH meter 0.2% Aminosoap, and 0.2% Amilite) on viruses of three was manually calibrated every 2 h during the preparation of completely different kinds; that is, HSV-1 and HSV-2 (Family test solutions. of Herpesviridae), IAV (Family of Orthomyxoviridae), and PV-1 (Family of Picornaviridae). Both HSVs and IAV are 2.3. Assay for Virucidal Activity. All the starting materials large enveloped viruses and need the cell nucleus for the were stored on ice prior to the virus inactivation exper- virus multiplication: the former has a double-stranded DNA iments. An excess volume of solutions was mixed with genome, and the latter has a segmented negative-stranded the virus stock so that both the pH and concentration of RNAgenome[16, 17]. In contrast to the viruses of these two the test solutions would not be affected: that is, a 190 μL virus families, PV-1 is a small nonenveloped virus carrying a positive-stranded RNA as a genome and replicates in the aliquot of the solutions received a 10 μL aliquot of virus 7 8 preparations (approximately 10 ∼10 plaque-forming units cytoplasm of the infected cells [18]. [PFU]/mL). This virus preparation was incubated at the Figure 1 shows the effects of these compounds on the indicated temperature for 5 min. After incubation, the test infectivity of HSV-1, when the virus was incubated at 30 C mixtures were chilled in ice-water bath, and, immediately, for 5 min. As a control, the virus was incubated in PBS aliquots of these virus samples were 100-fold diluted with and the relative residual infectivity for each compound was ice-cold Dulbecco’s phosphate-buffered saline (PBS) without calculated by dividing the number of the infectious virus in 2+ 2+ Ca and Mg containing 0.1% BSA for IAV or 0.5% the test solutions by that of the PBS control. As shown in FBS for other viruses to stop the virus inactivation. The Figure 1, HSV-1 was sensitive to these compounds except viruses were further diluted to yield virus counts suitable for Amisafe; the virus infectivity decreased below or near −5 for measurements, and the number of infectious virus in the detection level (10 of the control). In the case of the treated preparation was measured by a plaque assay on Amisafe, the decrease in the virus infectivity was marginally Vero (for HSV-1, HSV-2, and PV-1) or MDCK (for IAV) less than that incubated in the PBS. These results clearly show cells. There was little virus inactivation in PBS, and hence that arginine and amino acid derived compounds (except the amount of infectious virus in PBS was used as a control Amisafe) can efficiently inactivate HSV-1, an enveloped DNA count [14]. virus, under the experimental conditions. Although pH, ion Advances in Virology 3 0.1 0.1 0.01 0.01 0.001 0.001 0.0001 0.0001 0.00001 0.00001 Figure 1: Inactivation of HSV-1. The virus was incubated in the Figure 2: Inactivation of IAV. See the legend to Figure 1.Black and solution indicated below for 5 min at 30 C. Number of infectious white bars represent the results in the presence or absence of 0.1% viruswas determinedbyaplaqueassayonVerocells afterthe BSA. incubation and was normalized to that incubated in PBS. Arginine (0.5 M at pH 4.0 in 10 mM citrate), ButArg (0.5 M in pH 4.0 in 10 mM citrate), CAE (0.2% in 5 mM citrate and 0.15 M NaCl at of 0.1% BSA resulted in suppression, at least partially, of pH 4.8), Amisafe (0.2% in 5 mM citrate and 0.15 M NaCl at pH IAV inactivation by CAE, Amisafe, Amilite, BKC, SDS, and 4.8), Aminosoap (0.2% in 5 mM citrate at pH 7.0), Amilite (0.2% LS (black columns in Figure 2). This is unlikely due to the in 5 mM citrate at pH 8.0), BKC (0.1% in 5 mM citrate at pH 7.0), interaction of BSA with the virus, as it did not affect the virus SDS (0.2% in 5 mM citrate at pH 7.0), LS (0.2% in 5 mM citrate infectivity in PBS. It is most likely due to the ability of BSA to at pH 7.0), CHX (0.5% at pH 6.0), EtOH (70% in 5 mM citrate at bind fatty acids [19] and hence these compounds that have pH 7.0), PI (0.23% in 5 mM citrate at pH 3.5), NaOCl (100 ppm in 5 mM phosphate at pH 7.0), and PBS (pH 7.4). nonpolar moieties. The observed effects of these compounds on IAV suggest the potential mechanism of inactivation. Comparison of species, or ionic strength of the solution can affect the degree arginine, ButArg, CAE, Aminosoap, BKC, EtOH, PI, and of virus inactivation [14], we have shown before that HSV- NaOCl, which showed strong virucidal effects on this 1 is relatively stable above pH 4.8 and the effects of pH 4.0 virus, demonstrates no distinct pattern with compound alone are much weaker than the combined effects of the pH structure. Arginine and ButArg are known to suppress and arginine or ButArg [5, 6]. The results were compared protein-protein interactions, probably through their ability with commercial disinfectants (i.e., 0.1% BKC, 0.2% SDS, to interact with proteins [20–22]. CAE, Aminosoap, and 0.2% LS, 0.5% CHX, 70% EtOH, 0.23% PI, and 100 ppm BKC have surfactant properties. EtOH is an organic solvent. NaOCl). These commercial disinfectants also showed strong These compounds can interact with not only proteins, but virus inactivation on HSV-1 (Figure 1), although the effect also lipid membranes. PI and NaOCl are strong oxidants of PI may be entirely due to the low pH (i.e., pH 3.5). and can interact with most components of virus particles. Enveloped viruses are known to be sensitive to detergents, On the contrary, there appears to be a unique structure which can perturb and solubilize lipid bilayers in the viral pattern for less active compounds, that is, Amisafe, Amilite, envelope. In agreement with this, HSV-2, another human SDS, LS, and CHX. These are all detergents, meaning that herpesvirus of different serotype and pathogenicity, also detergent-like properties are insufficient for the effective showed a similar sensitivity profile to these amino acid virus inactivation. However, these detergents are effective derivatives and disinfectants (data not shown). against HSV-1, demonstrating that the virucidal effectiveness Next, we tested these compounds in Figure 1 against is virus dependent. In contrast to these enveloped viruses, IAV, an enveloped virus of another virus family. As shown poliovirus was resistant to inactivation by these amino in Figure 2 (white columns), the profile was quite different acid derived compounds, as only negligible degree of the from that observed for HSV-1 (and HSV-2). ButArg, CAE, inactivation was observed (Figure 3). Even combination of Aminosoap, BKC, EtOH, PI, and NaOCl reduced the IAV pH 4.0 and arginine or ButArg was insufficient. The lack −5 infectivity below the detection level (10 of the control). of sensitivity of PV-1 may suggest that inactivation of Arginine, Amisafe, Amilite, SDS, LS, and CHX showed a HSV-1 (Figure 1)and IAV(Figure 2) by these compounds varying degree of virucidal activities, with Amisafe being requires the presence of lipid envelope on the structure significantly less effective, similar to its effect on HSV-1, and of target viruses. It should be noteworthy that although with CHX being least effective (white columns in Figure 2). these compounds appear to act on lipids, it does not We have shown before that the pH profile of IAV inactivation necessarily mean solubilization of envelope lipid bilayer. The is complex [14] and, at least, a part of inactivation by low concentrations of the detergents used in these experiments pH samples (arginine, ButArg, and PI) is due to the pH, (0.2%) are below critical micelle concentrations (CMCs). but that the pH alone cannot explain the strong inactivation Although the CMCs of ionic detergents are generally affected by arginine and ButArg [5, 6]. Interestingly, the addition by the pH of detergent solutions, they are considered to Residual infectivity Arginine ButArg. CAE Amisafe Aminosoap Amilite BKC SDS LS CHX EtOH PI NaOCl PBS Residual infectivity Arginine ButArg. CAE Amisafe Aminosoap Amilite BKC SDS LS CHX EtOH PI NaOCl PBS 4 Advances in Virology 1 10 0.1 0.01 0.1 0.001 0.0001 0.01 0.00001 0.001 Figure 3: Inactivation of poliovirus. See the legend to Figure 1. 0.0001 0.00001 be in the range of ∼10 mM (approximately 0.4%) under 0 0.01 0.02 0.03 0.04 the experimental conditions. Below CMC, detergents are CAE (%) unlikely capable of destroying membrane structure. Figure 4: Inactivation of viruses by CAE. The viruses were Among these compounds, CAE and Aminosoap showed incubated at 20 C for 5 min in 10 mM citrate buffer (pH 5.0) a marked virucidal activity. Considering that CAE has been containing 0.15 M sodium chloride and varying concentrations of reported to have bacteriocidal activity [23], we further CAE. Number of infectious virus was determined by a plaque characterized the virucidal activity of CAE. assay after the incubation and was normalized to that incubated in PBS (pH 7.4). Circles ()for HSV-1; blackcircle(•)for HSV-2; 3.2. Characterization of Virucidal Activity of CAE. Figure 4 triangles () for IAV; squares ()for PV-1. shows the effects of CAE concentration on the inactivation of HSV-1, HSV-2, IAV, and PV-1. As CAE was more soluble required higher concentrations to achieve a similar level of at acidic pH, we used pH 5.0 in these experiments. This inactivation under these conditions. pH alone caused little inactivation for HSVs (less than 20% inactivation, if any) and PV-1 (almost no inactivation), but a 3.3. Cytotoxic Effects of the Reagents. The above three com- significant inactivation for IAV (50 ∼ 70% inactivation) [14]. pounds were then compared for cell toxicity. As shown in This is probably due to acid-induced conformational change Figure 6, BKC showed a strong cytocidal effect. When the of HA spike proteins present in IAV envelope [17]. Under monolayers of Vero cells were incubated in isotonic buffer the concentration range (0.001 to 0.04% CAE), PV-1 showed solution in the presence of various concentrations of CAE, only a marginal loss of the infectivity, while IAV showed a BKC, and SDS for 15 min at room temperature and numbers small, but significant (60% decrease at 0.04% CAE), decrease of dead and alive cells were determined by dye exclusion in infectivity with increasing CAE concentrations. In contrast test with trypan blue, BKC showed a drastic increase in the to these two viruses, both HSV-1 and HSV-2 showed a number of dead cells at the concentration above 0.002%. At sharp decrease in infectivity, leading to 10 -fold reduction the concentration required for the effective virucidal effect at 0.02% CAE for HSV-1 and ∼10 -fold reduction at 0.01% (0.003%), BKC killed more than 10% of the cells. In contrast CAE for HSV-2. Although both HSV-1 and HSV-2 belong to BKC, cell death by SDS and CAE occurred at 2.5- and to the same virus family, HSV-2 showed a significantly 10-fold higher concentrations, indicating their lower cell stronger inactivation than HSV-1 at low CAE concentrations. toxicity. It should be noted that SDS solubilized plasma membrane of the cells at the cytotoxic concentrations while This virucidal activity of CAE was slightly, but significantly, CAE or BKC did not (data not shown), leading to a possible enhanced by lowering the pH of the test solutions to 4.0 or 4.5; for example, the inactivation of HSV-1 or HSV-2 at underestimation of the fraction of dead cells in the SDS- treated dishes. The results suggest that at 0.01% CAE, about 0.005% CAE was only marginal at pH 5.0, but was enhanced 2% of the cells were killed by the compound (Figure 6), while 10-fold (for HSV-1) or 100-fold (for HSV-2) at pH 4.0 at the same CAE concentration. more than 99% of the virus was inactivated (Figure 5). To further characterize the virucidal effects of CAE, it was compared with SDS, an anionic detergent, and BKC, 3.4. Inhibition of Virus Multiplication by CAE. The results in a cationic detergent, at neutral pH. Figure 5 shows such Figures 1–5 show in vitro virus inactivation. Whether the a comparison of virucidal activities against HSV-2. Both same compounds can yield a reduced virus multiplication BKCand SDSshowedmarkedeffects (more than two-log in in vivo experiments was then examined by treating the reduction) on HSV-2 infectivity even at low concentrations, infected cells with them. Figure 7 shows the results of CAE for example, at 0.002% and 0.003%, respectively, while CAE and BKC on the relative virus yields of HSV-1 (circle) or Residual infectivity Arginine ButArg. CAE Amisafe Aminosoap Amilite BKC SDS PBS Relative virus yield Advances in Virology 5 0.1 0.1 0.01 0.01 0.001 0.0001 0 0.005 0.01 0.001 Reagent (%) Figure 5: Effects of CAE, BKC, and SDS on the infectivity of HSV-2. HSV-2 was incubated in PBS (pH 7.4) containing varying 0.0001 concentrations of each compound at 30 C for 5 min. The amounts 0 0.005 0.01 of infectious progeny viruses were determined by a plaque assay Reagent (%) after the incubation and were normalized to that incubated in PBS (pH 7.4). Circles () for CAE; triangles ()for BKC; squares() for SDS. Figure 7: Effect of CAE and BKC on the virus multiplication. Confluent monolayers of HEp-2 cells were infected with HSV-1 (, •) at an MOI of 14 or PV-1 (, ) at an MOI of 9. The infected cells were incubated for overnight in the medium containing varying concentrations of CAE (, )orBKC (•, )at37 Cfor HSV-1or at 35.5 C for PV-1. At the end of infection (20 h after infection for HSV-1 and 16 h after infection for PV-1), the amounts of infectious 0.1 progeny viruses were determined and were normalized to the virus yield in the absence of these compounds. 0.01 inhibit the multiplication of two viruses widely different in the way the replication and transcription of the viral genome occur (i.e., in the nucleus or in the cytoplasm of the infected 0.001 cells). It is interesting to note that BKC and CAE, which were 0 0.01 0.02 both ineffective even at 0.1 and 0.2% in inactivating PV-1, Reagent (%) can suppress the growth of the same virus in the infected Figure 6: Effect of CAE, BKC, and SDS on the cell viability. cells. It is evident that the decreased virus yield in infected Confluent monolayers of Vero cells were incubated in 0.85% cells for PV-1 (and perhaps HSV-1) is not simply due to the sodium chloride solution (pH 5.5) containing 20 mM MES and virus inactivation. varying concentrations of each compound at room temperature Generally, virus-inactivating agents (disinfectants) show for 15 min. The treated cells were trypsinized to obtain a single toxicity to cells and tissues, limiting their applications. cell suspension and the amounts of the live and dead cells in each We have undertaken a systematic screening of amino acid culture were determined by a dye exclusion test with trypan blue. derived compounds in search for effective, nontoxic virus- Circles () for CAE; triangles ()for BKC; squares() for SDS. inactivating agents and found that, against HSV-1 and HSV- 2, CAE has notable activities to inactivate the infectivity PV-1 (triangle), when the virus-infected cells were incubated of extracellular virus particles and to suppress the virus in the medium containing varying concentration of CAE multiplication in the infected cells at the concentration with (open symbol) or BKC (closed symbol): note that the highest tolerable cytotoxic effect. These results may support a future concentrations used are still below the concentrations used potential application of CAE as a therapeutic or preventive for virus inactivation. The multiplication of these two viruses medicine against HSV superficial infection at body surface, was much more strongly suppressed by BKC than CAE. such as herpetic keratitis and genital herpes. However, their effects on these two viruses were different. Namely, CAE suppressed more strongly the virus yield of PV-1 than HSV-1, opposite to BKC. Comparison of the Conflict of Interests results in Figure 6 suggests that the suppression of these virus multiplications by CEA or BKC was not due to the death of The authors do not have commercial or other associations the infected cells. These results show that both BKC and CEA that might pose a conflict of interests. Residual infectivity Fraction of dead cells Relative virus yields 6 Advances in Virology Acknowledgments [16] B. Roizman and D. M. 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