Comparative Virucidal Activities of Essential Oils and Alcohol-Based Solutions against Enveloped Virus Surrogates: In Vitro and In Silico Analyses

The large-scale use of alcohol (OH)-based disinfectants to control pathogenic viruses is of great concern because of their side effects on humans and harmful impact on the environment. There is an urgent need to develop safe and environmentally friendly disinfectants. Essential oils (EOs) are generally recognized as safe (GRAS) by the FDA, and many exhibit strong antiviral efficacy against pathogenic human enveloped viruses. The present study investigated the virucidal disinfectant activity of solutions containing EO and OH against DENV-2 and CHIKV, which were used as surrogate viruses for human pathogenic enveloped viruses. The quantitative suspension test was used. A solution containing 12% EO + 10% OH reduced > 4.0 log10 TCID50 (100% reduction) of both viruses within 1 min of exposure. In addition, solutions containing 12% EO and 3% EO without OH reduced > 4.0 log10 TCID50 of both viruses after 10 min and 30 min of exposure, respectively. The binding affinities of 42 EO compounds and viral envelope proteins were investigated through docking analyses. Sesquiterpene showed the highest binding affinities (from −6.7 to −8.0 kcal/mol) with DENV-2 E and CHIKV E1-E2-E3 proteins. The data provide a first step toward defining the potential of EOs as disinfectants.


Introduction
Enveloped RNA viruses such as coronavirus, influenza A (HIN1) virus, and Ebola virus are responsible for pandemics and epidemics, which are transmitted primarily through close person-to-person contact as well as through aerosolized respiratory droplets [1,2]. A susceptible person can also be infected by indirect transmission by self-inoculation through the mucous membranes of the nose and mouth by touching contaminated surfaces. Viruses can persist for hours or even days on inanimate surfaces [1]. Therefore, the use of disinfecting agents for surface cleaning and personal care is one of the first-line strategies to limit virus transmission during an epidemic [3].
The World Health Organization recommends alcohol (OH)-based hand sanitizers to control the transmission of human pathogenic enveloped viruses [4]. Generally, OH-based virucidal disinfectants contain high concentrations of ethanol (80% v/v) or isopropanol (70% v/v) or a combination of these [5,6]. Because of their lipophilicity, OHs damage the phospholipid membrane of viruses by the delipidation and denaturation of proteins. Although OH-based disinfectants exhibit strong virucidal activity, they have limitations and their excessive use can be a threat to living beings [7][8][9]. OHs are flammable liquids, and prolonged exposure to ethanol causes skin and eye irritation; alcohol evaporates Table 1 shows the solutions tested for virucidal disinfectant activity. A single EO blend was used, which contained pure EOs from seven Colombian aromatic plants. In addition, an OH preparation was used, which contained ethanol (70%) and a mixture (25%) of isopropanol and glycerol. Eight solutions containing EO (3%, 6%, and 12%) combined or not with the OH preparation (1%, 5% and 10%) were analyzed. The cytotoxicity assay revealed that none of the test solutions were cytotoxic to Vero cells ( Table 2). The cell viability ranged from 80% up to 100%, relative to untreated cells, after incubation of the cells with the lowest dilution (1:10) of each solution.  Acetic acid (5%) was used as a virucidal agent. Data are averages ± SDs from three independent assays in triplicate.

Virucidal Disinfectant Activity of the Test Solutions
The quantitative suspension test was used to evaluate the antiviral disinfectant activity following the German DVV/RKI guideline [19], and limits were 3.8-5 ± 1.2 and 5.3 ± 0.55 TCID 50 (log10) per mL of DENV-2 and CHIKV, respectively (Table 3). A reduction factor of 4-log10 was the cutoff value for disinfectant activity [19]. First, we evaluated the activity of solutions containing 12% EO combined or not with OH within 1 min of exposure ( Figure 1A). The solution containing 12% EO without OH (12EO) was sufficient to achieve a 3.9-log10 (81.6%) reduction in the DENV-2 titer, but was insufficient to reduce the CHIKV titer. The addition of 1% (12EO + 1OH solution) and 5% (12EO + 5OH solution) OH increased the reduction of DENV-2 to >4-log10 (100%), whereas the addition of 10% OH (12EO + 10OH solution) was required to achieve a 4-log10 (100%) reduction of CHIKV. Next, we evaluated solutions containing EO at concentrations lower than 12% combined with 10% OH after 1 min of exposure ( Figure 1B). A reduction of 4-log10 of DENV-2, but not CHIKV, was achieved with the 6% EO + 10% OH (6EO + 10OH) solution, whereas the reduction of both viruses was not observed with the 3% EO + 10% OH (3EO − 10OH) solution. Solutions containing 3% EO without OH (3EO) and 10% OH without EO (10OH) did not show a virucidal effect against either virus after 1 min of exposure. As the solutions containing 12% EO and 3% EO without OH did not show disinfectant effects against DENV-2 and CHIKV after 1 min of exposure, we assessed the activity of these solutions by increasing the exposure time in four intervals (Table 4). For the 12% EO solution (12EO), a reduction of >4-log10 (100%) of DENV-2 was achieved after 5 min of exposure, whereas a 100% reduction of CHIKV was achieved after 10 min of exposure. For As the solutions containing 12% EO and 3% EO without OH did not show disinfectant effects against DENV-2 and CHIKV after 1 min of exposure, we assessed the activity of these solutions by increasing the exposure time in four intervals (Table 4). For the 12% EO solution (12EO), a reduction of >4-log10 (100%) of DENV-2 was achieved after 5 min of exposure, whereas a 100% reduction of CHIKV was achieved after 10 min of exposure. For the 3% EO solution, an exposure time of 30 min was required to achieve a 100% reduction in DENV-2 and CHIKV.  Table 5 presents the linear retention indices and relative amounts of compounds in order of their elution on the DB-5MS column. A chromatogram of the EO blend is presented ( Figure S1, Supplementary Materials). Forty-two compounds were identified. Monoterpene alcohols (52%) and aldehydes (23.8%) were the most abundant terpenes, especially geraniol (35.4%), citronellal (22.6%), and citronellol (14.1%), followed by monoterpene acetates (7.2%) and hydrocarbons (4.2%). Sesquiterpenoids were identified in low concentration (8.4%) and sesquiterpene hydrocarbons (5.7%), mostly germacrene D and δ-cadinene, were in higher concentration than oxygenated sesquiterpenes.  [20]; b [21]; c [22]; d [23]. * Use of standard compounds.
The CHIKV particle has a capsid surrounded by a lipid envelope, which contains the E1-E2-E3 glycoprotein complex [26]. The forty-two compounds identified in the EO blend were subjected to molecular docking against E1-E2-E3. Table S1 presents the AutoDock Vina binding energies. Eight of the ten oxygenated sesquiterpenes identified in the EO blend bound to E1-E2-E3, and α-cadinol, α-eudesmol, and caryophyllene oxide exhibited the lowest binding energies (−6.50 to −6.70 kcal/mol) followed by patchoulol, germacrene D-4-ol, and epi-α-muurolol (−6.37 to −6.45 kcal/mol). In addition, nine of the ten sesquiterpenes hydrocarbons bound to the E1-E2-E3 complex, and α-guaiene, α-humulene, and trans-β-caryophyllene exhibited the lowest binding energies (−6.32 to −6.38 kcal/mol) followed by α-bulnesene, germacrene D, and δ-cadinene (−6.25 to −6.26 kcal/mol). The sesquiterpenes were accommodated in two consensus sites corresponding to a pocket in the domain II of the E1 protein (six sesquiterpenes) and a pocket in the β-ribbon connector of E2 protein (eleven sesquiterpenes). All EO compounds formed hydrophobic bonds with amino acid residues and five of the top compounds formed hydrogen bonds with amino acids lining the pocket (Figure 3). Table 7 presents the EO compounds with the lowest binding energy with the CHIKV E1-E2-E3 complex.  The twelve compounds with a strong binding energy (−7.03 to −8.61 kcal/mol) an compounds with a weak binding energy (−6.0 to −6.7 kcal/mol) were reported in a [25].
The CHIKV particle has a capsid surrounded by a lipid envelope, whic E1-E2-E3 glycoprotein complex [26]. The forty-two compounds identified in were subjected to molecular docking against E1-E2-E3. Table S1   The twelve compounds with a strong binding energy (−7.03 to −8.61 kcal/mol) an compounds with a weak binding energy (−6.0 to −6.7 kcal/mol) were reported in a [25].
The CHIKV particle has a capsid surrounded by a lipid envelope, whi E1-E2-E3 glycoprotein complex [26]. The forty-two compounds identified i were subjected to molecular docking against E1-E2-E3.  The twelve compounds with a strong binding energy (−7.03 to −8.61 kcal/mol) an compounds with a weak binding energy (−6.0 to −6.7 kcal/mol) were reported in a [25].

Discussion
Cleaning virus-contaminated hands and surfaces is essential for infection control and viral disease prevention [4]. OH-based solutions are utilized as disinfectants to control the transmission of human pathogenic viruses. However, frequent and prolonged use of OHbased disinfectants may be harmful to health and the environment [7][8][9]. EOs in the form of natural products are generally recognized as safe (GRAS) by the FDA (Food

Discussion
Cleaning virus-contaminated hands and surfaces is essential for infection control and viral disease prevention [4]. OH-based solutions are utilized as disinfectants to control the transmission of human pathogenic viruses. However, frequent and prolonged use of OHbased disinfectants may be harmful to health and the environment [7][8][9]

Discussion
Cleaning virus-contaminated hands and surfaces is essential for infection control and viral disease prevention [4]. OH-based solutions are utilized as disinfectants to control the transmission of human pathogenic viruses. However, frequent and prolonged use of OHbased disinfectants may be harmful to health and the environment [7][8][9]. EOs in the form of natural products are generally recognized as safe (GRAS) by the FDA (Food

Discussion
Cleaning virus-contaminated hands and surfaces is essential for infection control and viral disease prevention [4]. OH-based solutions are utilized as disinfectants to control the transmission of human pathogenic viruses. However, frequent and prolonged use of OHbased disinfectants may be harmful to health and the environment [7][8][9]. EOs in the form of natural products are generally recognized as safe (GRAS) by the FDA (Food  trans-β-Caryophyllene E2, β-ribbon connector Ile136, Phe141, Arg144 −6.32 ± 0.1

Discussion
Cleaning virus-contaminated hands and surfaces is essential for infection control and viral disease prevention [4]. OH-based solutions are utilized as disinfectants to control the transmission of human pathogenic viruses. However, frequent and prolonged use of OHbased disinfectants may be harmful to health and the environment [7][8][9]. EOs in the form of natural products are generally recognized as safe (GRAS) by the FDA (Food and Drug E2, β-ribbon connector Ile136, Phe141 −6.37 ± 0.3 trans-β-Caryophyllene E2, β-ribbon connector Ile136, Phe141, Arg144 −6.32 ± 0.1

Discussion
Cleaning virus-contaminated hands and surfaces is essential for infection control and viral disease prevention [4]. OH-based solutions are utilized as disinfectants to control the transmission of human pathogenic viruses. However, frequent and prolonged use of OHbased disinfectants may be harmful to health and the environment [7][8][9]. EOs in the form of natural products are generally recognized as safe (GRAS) by the FDA (Food and Drug E2, β-ribbon connector Ile136, Phe141, Arg144 −6.32 ± 0.1

Discussion
Cleaning virus-contaminated hands and surfaces is essential for infection control and viral disease prevention [4]. OH-based solutions are utilized as disinfectants to control the transmission of human pathogenic viruses. However, frequent and prolonged use of OH-based disinfectants may be harmful to health and the environment [7][8][9]. EOs in the form of natural products are generally recognized as safe (GRAS) by the FDA (Food and Drug Administration, Silver Spring, MD, USA), and their use is permitted [27]. Many studies have explored using EOs as potential antibacterial and antifungal alternatives to commercial disinfectants [14,28]. In contrast, scientific evidence supporting the potential of EOs as disinfectants against enveloped viruses is very limited. Our study focused on enveloped viruses; studies show that enveloped viruses tend to infect more host species and are more likely to be pandemic than non-enveloped viruses [1,29].
The present study evaluated the virucidal disinfectant activity of solutions containing EO and OH against two surrogate viruses for pathogenic enveloped viruses. The results show that a solution of 12% EO combined with 10% OH reduced up to >4.0 log10 TCID 50 (100% reduction) of both viruses within 1 min of exposure. In addition, the solutions containing EO without OH also exhibited virucidal action (100% reduction) against both viruses after 10 min (12% EO) and 30 min (3% EO) of exposure. We did not observe a 100% reduction in either virus with the 10% OH solution, but when combined with 12% EO, a strong virucidal activity was observed. It appears that low concentrations of EO and OH are insufficient to inactivate human pathogenic enveloped viruses. Romeo et al. [18] did not observe virucidal activity of a formulation containing 3.3% EO (Melaleuca alternifolia) combined with 5.3% ethanol against the coronavirus HCov-OC43 after 30 min of exposure.
To evaluate the virucidal disinfectant activity, we used two enveloped viruses, which differ in the lipid content [30,31] and protein structure [24,26] that comprise the viral envelope. The results indicated that DENV-2 was more sensitive to the action of test solutions than CHIKV. We hypothesized that differences in the viral envelope structure and its hydrophobic/hydrophilic nature might explain the variation in sensitivity. The DENV-2 particle assembles and buds into the endoplasmic reticulum of the infected cells where the envelope is formed. The envelope has 90 head-to-tail dimers of the E protein organized in a herringbone, with the M protein bound at the dimer interface [32]. On the other hand, CHIKV assembles and budding occurs at the cytoplasmic membrane, and the viral envelope comprises the E1 and E2 glycoproteins and a peptide (E3) arranged in trimers to make 80 E1/E2 spikes [27]. A recent study [18] showed differences in the sensitivity of enveloped viruses (human and feline coronaviruses) to treatment with a mixture of tea tree oil and ethanol.
Enveloped viruses enter host cells primarily via endocytosis following attachment to a cellular receptor [2,29,33]. Upon attachment, viruses are engulfed into endosomes where the low pH triggers conformational changes of the envelope proteins to drive fusion of the viral envelope and endosomal membrane. The viral envelope plays an important role in the membrane fusion process [33], and envelope proteins are potential extracellular drug targets with multiple strategies to inhibit entry of the virus into host cells [34]. Studies suggest that EOs could cause the morphological alteration of the viral particle by destroying the viral envelope through interactions between their terpene constituents and viral proteins [11,13]. In silico and in vitro evidence suggests that sesquiterpene hydrocarbons and oxygenated monoterpenes in specific ratios may account for the antiviral action of the EOs [11][12][13]. Recently, we documented a variation in the anti-DENV effect related to variation in oxygenated monoterpene content [25]. We also documented [35] a better in vitro anti-DENV effect of trans-β-caryophyllene and geranyl acetate compared to p-cymene, limonene, and neral, all of which were identified in the test EO blend.
We performed a primary docking analysis to describe the interactions between the 42 compounds of the EO blend and the envelope proteins of DENV-2 (E) and CHIKV (E1-E2-E3). As in a previous study [25], in the present study, we again found sesquiterpene hydrocarbons and oxygenated monoterpenes showing good binding affinities (−6.7 to −8.6 kcal/mol) with the DENV-2 E protein. These terpenes were accommodated in the βOG pocket and molecules that dock this pocket can block the conformational change of the E protein required for the fusion process [36]. As for CHIKV, seventeen EO compounds docked the E1-E2-E3 glycoprotein complex. Some bound to the E2 protein in a pocket of the β-ribbon connector peptides, which play a role during virus entry, helping to trigger E1 conformational changes during the fusion process [37]. Other EO compounds bound to the E1 protein of CHIKV near the hydrophobic fusion loop, which mediates membrane fusion [37]. According to the docking score values, EO compounds exhibited better binding affinities against DENV-2 than against CHIKV, which could partly explain the differences in sensitivity to the test solutions revealed in the virucidal disinfectant assays.
Little is known about the specific mechanism of action of EOs against enveloped viruses. Mechanisms other than alterations of the envelope protein structure have been proposed [11][12][13]. Being lipophilic, EOs can penetrate the viral envelope and cause membrane disintegration; they can cause viral expansion, which interferes with the attachment process by which viruses gain entry into host cells; moreover, EO components can inhibit host lipid metabolism pathways, which are crucial to ensure the availability of lipids to complete the assembly of new enveloped virions. On the other hand, OH causes protein denaturation and the disruption of the viral envelope [5]. Ethanol (95%, v/v) has broader and stronger virucidal activity than propanols (75% v/v); isopropanol, due to its lipophilic nature, interacts favorably with viral envelopes, and glycerol (80% v/v) and glycerol derivatives have been described as virucidal agents against enveloped viruses [38,39]. We hypothesize that the EO and OH mechanisms mentioned here could be involved in the strong virucidal disinfectant activity of the 12% EO + 10% OH solution.
The results of this study demonstrate that EO alone not only has disinfectant activity, but also shows synergistic activity with OH against two enveloped viruses. This synergistic activity may involve all of the aforementioned mechanisms of action of EOs. Further analysis is needed to investigate the contribution of each EO compound and their additive, synergistic, or antagonistic effects on the disinfectant action of a pure EO.

Plant Material and EO Blend
Pure EOs from seven aromatic plants grown in Colombia were used to prepare an EO blend. Then, a stock solution (6 × 10 6 µg/mL) of the EO blend was prepared in DMSO and it was used to prepare the test solutions for analyses of the disinfectant activity (Table 1) Colombia). The taxonomic identification of these plants was performed at the Colombian National Herbarium (Bogotá, Colombia), where their vouchers were placed. EOs were obtained through the hydrodistillation (2 h) of plant leaves and stems on a Clevenger apparatus as described elsewhere [40,41].

Chemical Composition of the EO Blend
The analysis of the EO blend was performed by gas chromatography using mass spectrometric (GC/MS) and flame ionization detection (GC/FID) systems. Previous studies described the conditions of the process and data analysis [25,42,43]. Before the analysis, the EO blend was dissolved in dichloromethane (1 mL). n-Tetradecane (0.5 µL) was added as an internal standard. The injection volume was 2 µL in split mode (30:1). A 6890 Plus Gas Chromatograph (Agilent Technologies, AT, Palo Alto, CA, USA) equipped with a mass selective detector MSD 5975 (Electron ionization, EI, 70 eV), (AT, Palo Alto, CA, USA), a 7863 automatic injector, and an MSChemStation G1701DA data system (AT, Palo Alto, CA, USA) were used. The identification of EO compounds was accomplished by the comparison of their linear retention indices (LRIs) with those of standard compounds, and by the comparison of their mass spectral fragmentation patterns with those described in the literature and databases [20][21][22][23][24].

Preparation of the Test Solutions
Pure EOs from seven aromatic plant species were mixed in various proportions to obtain an EO blend using dimethyl sulfoxide (DMSO) as the solvent. The EO blend was mixed with the desired amount of an OH mixture (ethanol ca. 70%; isopropanol + glycerol ca. 2.5%) in a glass vial to give five different percentage ratios of EO/OH. Each solution was stirred using a vortex mixer until complete mixing took place. In addition, the EO blend was diluted to give solutions of 12% and 3%, and the OH mixture was diluted in water to prepare a 10% solution.

Cells and Viruses
Vero cells (African green monkey kidney cells; CCL-81™. ATCC, Manassas, VA, USA) were cultured in minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic at 37 • C in a humidified atmosphere of 5% CO 2 . DENV-2 NGC (CDC, San Juan, Puerto Rico) was propagated in C6/36 Aedes albopictus cells (Pedro Kourí Institute for Tropical Medicine, La Habana, Cuba). CHIKV, a local strain isolated from a patient in our laboratory [44], was propagated in Vero cells. Both viruses were titrated using a protocol of the median tissue culture infectious dose (TCID 50 )-Spearman Karber assay [45].

Cytotoxicity Controls
As EO and OH can cause cytotoxic effects, the test solutions were first evaluated in Vero cells without the addition of virus. Briefly, the test solution was serially diluted, and an aliquot was added to cells seeded in 96-well plates. Following 1 h of incubation at 37 • C, the solution was discarded by washing and the cells were overlaid with fresh culture medium and incubated for 72 h at 37 • C and 5% CO 2 . Next, the cell viability was determined by staining with crystal violet, as in a previous study [25]. Briefly, 100 µL of 0.05% crystal violet solution was added to cells for 20 min at room temperature. After washing, the plates were aspirated and allowed to air dry at room temperature, and 200 µL of methanol was added to each well for 20 min. The optical density at 570 nm in each well was measured on a microplate reader (570 nm) to quantify crystal violet staining.

Evaluation of Virucidal Disinfectant Activity
The quantitative suspension test was used following the German DVV/RKI guideline [13,18]. The test was performed in five intervals (1, 5, 10, 20, and 30 min) of exposure of the virus with the test solution with fixed amounts of DENV-2 (8.4 log10 TCID 50 /mL) and CHIKV (7.8 log10 TCID 50 /mL). Briefly, 10 µL of a virus preparation was mixed with 80 µL of solution and 10 µL of water, and a virus control with 90 µL of water without test solution was included. At the end of the exposure times, 900 µL of ice-cold culture medium was added to each mixture and immediately diluted 10-fold to determine viral infectivity using end-point dilution titration. Vero cells were seeded in 96-well plates for 24 h at 37 • C under 5% CO 2 and infected with serial dilutions of treated DENV-2 and treated CHIKV in triplicate on a logarithmic scale at base 10. Noninfected cells were included as controls. The plates were incubated at 37 • C and 5% CO 2 for five days. After washing, the plates were aspirated and allowed to air dry at room temperature, and the crystal violet dye uptake was determined as described above. The quantity of virus was calculated as TCID 50 (log10) per milliliter by the Spearman-Karber method [45].

Docking Analysis
Three-dimensional structures of DENV-2 E protein (PDB ID: 10AN) and the CHIKV E1-E2-E3 complex (PDB ID: 3N42) were downloaded from the Protein Data Bank. Structures of chemical constituents of the EO blend were retrieved from the PubChem (https://pubchem. ncbi.nlm.nih.gov/ (accessed on 13 April 2023)) database. The preparation of the target and ligands and molecular docking analyses were carried out using AutoDock Vina (Version 1.5.6, La Jolla, CA, USA), as described in a previous study [25]. The optimized protein structure was saved in the PDBQT file format for docking analysis. Default parameters were used, and the search exhaustiveness parameter was set to 100. For each ligand, 27 docked conformations were generated using global docking simulations. Three simulations were performed for each ligand-protein pair using seeds 6, 12, and 18. The average docking scores for each protein approximated the binding free energy. Discovery Studio Visualizer v21.1.0.20298 was used to view the ligand-protein interactions.

Statistical Analyses
A one-way ANOVA and a Tukey-Kramer post hoc test of viral titer values were used to compare the virucidal effect of each test solution, adopting a significance level of 0.05. The data were analyzed using GraphPad Prism software (version 8.0, San Diego, CA, USA).

Conclusions
The inadequate and inappropriate use of OH-based and other disinfectants has been associated with harmful effects on humans and the environment. There is an urgent need to develop safe and environmentally friendly disinfectants to minimize adverse effects. The data from this study provide a first step in defining the potential utility of EOs as disinfectants to control the transmission of human pathogenic enveloped viruses. We conclude that a solution containing 12% of a mixture of seven EOs and 10% of a mixture of OHs (ethanol, isopropanol, and glycerol) is highly effective for the inactivation of DENV-2 and CHIKV, which could be extended to enveloped viruses of similar structure that are transmitted person-to-person. In addition to reducing virus titers to 100%, the solution acts within one minute, making it practical for use in environments where rapid disinfection is needed. The hydrocarbon sesquiterpenes, oxygenated sesquiterpenes, and oxygenated monoterpenes present in the EO blend showed binding affinities for DENV-2 and CHIKV envelope proteins, suggesting that these types of terpenes could act as inhibitors of virus adsorption and entry into host cells. Further analysis is needed to better define the potential of EOs as virucidal disinfectant alternatives to commercial OH-based disinfectants.