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Protective essential oil attenuates influenza virus infection: An in vitro study in MDCK cells
© Wu et al; licensee BioMed Central Ltd. 2010
Received: 10 September 2010
Accepted: 15 November 2010
Published: 15 November 2010
Influenza is a significant cause of morbidity and mortality. The recent pandemic of a novel H1N1 influenza virus has stressed the importance of the search for effective treatments for this disease. Essential oils from aromatic plants have been used for a wide variety of applications, such as personal hygiene, therapeutic massage and even medical practice. In this paper, we investigate the potential role of an essential oil in antiviral activity.
We studied a commercial essential oil blend, On Guard™, and evaluated its ability in modulating influenza virus, A/PR8/34 (PR8), infection in Madin-Darby canine kidney (MDCK) cells. Influenza virus was first incubated with the essential oil and infectivity in MDCK cells was quantified by fluorescent focus assay (FFA). In order to determine the mechanism of effects of essential oil in viral infection inhibition, we measured hemagglutination (HA) activity, binding and internalization of untreated and oil-treated virus in MDCK cells by flow cytometry and immunofluorescence microscopy. In addition, the effect of oil treatment on viral transcription and translation were assayed by relative end-point RT-PCR and western blot analysis.
Influenza virus infectivity was suppressed by essential oil treatment in a dose-dependent manner; the number of nascent viral particles released from MDCK cells was reduced by 90% and by 40% when virus was treated with 1:4,000 and 1:6,000 dilutions of the oil, respectively. Oil treatment of the virus also decreased direct infection of the cells as the number of infected MDCK cells decreased by 90% and 45% when virus was treated with 1:2,000 and 1:3,000 dilutions of the oil, respectively. This was not due to a decrease in HA activity, as HA was preserved despite oil treatment. In addition, oil treatment did not affect virus binding or internalization in MDCK cells. These effects did not appear to be due to cytotoxicity of the oil as MDCK cell viability was only seen with concentrations of oil that were 2 to 6 times greater than the doses that inhibited viral infectivity. RT-PCR and western blotting demonstrated that oil treatment of the virus inhibited viral NP and NS1 protein, but not mRNA expression.
An essential oil blend significantly attenuates influenza virus PR8 infectivity in vitro without affecting viral binding or cellular internalization in MDCK cells. Oil treated virus continued to express viral mRNAs but had minimal expression of viral proteins, suggesting that the antiviral effect may be due to inhibition of viral protein translation.
The recent pandemic of a novel H1N1 influenza emphasizes the urgency of identifying effective approaches to prevent viral infection. Between 1990 and 1999 in the United States, non-pandemic influenza A virus (IAV) infected 5-20% of people and caused approximately 36,000 deaths and 226,000 hospitalizations annually [1, 2]. IAV infection is initiated with a binding of the viral hemagglutinin (HA) to sialic acid on the cell surface; and virus particles are internalized through receptor-mediated endocytosis. While in the endosome, viral HA protein is activated and the virus fuses with endosomal membranes. After fusion, IAV shuts off host cell protein synthesis and cell replication. As a result, infected cells die by apoptosis or cytolysis.
Essential oils have been used for aromatherapy, massage therapy, emotional health, personal care, nutritional supplements, or cleaning for many years. The modern use of essential oils has grown rapidly as health scientists and medical practitioners have found scientific evidence for the benefits of this therapy. In Japan, Perillae Herba (a leaf of Perilla frutescens) has been prescribed to treat depression. It has been shown that l-Perillaldehyde, a major component in the essential oil containing in Perillae Herba, is responsible for the antidepressant-like activity through stimulation of the olfactory nerve . Essential oil from Boswellia carteri and Boswellia serrata have been used for the treatment of rheumatoid arthritis and other inflammatory diseases in traditional medicine for many years [4, 5]. Studies showed that frankincense oil derived from Boswellia species possess anti-inflammatory activity though inhibition of immune cytokines production and leukocyte infiltration [6–8]. In addition, many other essential oils used in aromatherapy have medicinal properties including antiseptic properties , and mood enhancing effects [10, 11].
Distinctive chemical components of plants protect them from insects, bacteria or viruses that cause diseases. Essential oils prepared from plants, therefore, might be effective in protecting humans from viral infection. In addition to their intrinsic benefits to plants and as fragrances for people, essential oils have been used throughout history in many cultures for their medicinal and therapeutic benefits. They were first used in ancient Egypt for treatment of various illnesses and other physical and spiritual needs. Borrowing from the Egyptians, the Greeks, Romans, Indians, Persians, as well as Chinese began to refine distillation methods for extracting oils from aromatic plants and have used them extensively in medical practice for diverse purposes, such as promoting wellness, enhancing personal hygiene, and in therapeutic massage and aromatherapy. They have also been used as a beauty treatment, in food preparation, and in religious ceremonies.
In this study, we evaluated the effect of a commercially available essential oil blend, On Guard™, on influenza virus A/PR/8/34 (PR8) infection in Madin-Darby canine kidney (MDCK) cells. This oil blend combines a mixture of wild orange, clove, cinnamon, eucalyptus and rosemary. The mechanism of the oil-mediated inhibition of viral infectivity was also investigated.
Reagents and chemicals
Cell culture medium MEM and DMEM/F12 (1:1), fetal calf serum (FCS), and penicillin-streptomycin were purchased from Invitrogen (Carlsbad, CA). Trypan blue was purchased from Sigma (St. Louis, MO). On Guard™ protective blend oil was obtained from dōTERRA International (Orem, UT). Mouse anti-IAV nucleoprotein (NP) monoclonal antibody was purchased from Abcam (Cambridge, MA). Alexa Fluro 488-conjugated goat anti-mouse IgG was purchased from Molecular Probes (Eugene, OR). Goat anti-IAV NS1 polyclonal antibody was purchased from Santa Cruz (Santa Cruz, CA).
Preparation of influenza virus stock and cell culture
Influenza A/PR/8/34 (PR8), a laboratory adapted H1N1 IAV strain, was passaged in MDCK cells. MDCK cells were cultured in DMEM supplemented with 10% FCS. Viruses were grown in MDCK cells in DMEM/F12 with ITS+ (containing insulin, human transferrin, and selenous acid; BD Biosciences, Franklin Lakes, NJ) and trypsin (0.5 μg/ml), harvested at 72 h postinfection and titered by plaque assay in MDCK cells. There was no detectable endotoxin in the final viral preparations used in the experiments as determined by limulus amebocyte lysate assay (Cambrex, Walkersville, MD). The assay has a detection limit of 0.1 EU/ml or approximately 20 pg/ml lipopolysaccharide (LPS).
For essential oil-treated influenza virus, PR8 virus was mixed with serial dilutions of the essential oil and incubated at room temperature for 2 h. PR8 virus mixed and incubated in 1-1,000 dilutions of canola oil was used as a control. Oil-treated and untreated IAV was stored at -80°C until use.
Fluorescent focus assay (FFA)
To determine the release of IAV infectious particles from oil-treated and untreated IAV-infected MDCK cells, cells were grown to confluence in 24-well plates and oil-treated and untreated PR8 virus was added at the multiplicity of infection (MOI) of 1 and incubated at 37°C in a cell incubator. At 48 h after infection, 50 μl aliquots of cell culture supernatants were collected and transferred to 96-well plates containing confluent MDCK cells. The supernatants were serially diluted with PBS containing 0.6 mM CaCl2 and 0.5 mM MgCl2 (CaMg-PBS) and incubated with the MDCK cells for 45 minutes at 37°C, followed by washing of the cells three times in serum-free DMEM/F12 with ITS+ containing 1% penicillin and streptomycin. The cells were maintained in DMEM/F12 with ITS+.
After 6 h at 37°C, the MDCK monolayers were subsequently washed three times with PBS and fixed with 4% paraformaldehyde for 10 min at 4°C followed by permeabilization with 2% Triton X-100 for 10 min in room temperature. The cell monolayers were then labeled by incubating with a monoclonal antibody (Abcam, Cambridge, MA) against the IAV NP in Staining Buffer (PBS with 0.1%BSA, 1% heat-inactivated human serum, 0.02%NaN3) for 30 min at 4°C. Following washes with PBS, Alexa Fluor 488-conjugated goat anti-mouse IgG was added to detect antibody binding to the IAV NPs. Initially, various dilutions of virus were used to find the dose yielding ~50 fluorescent foci per high powered (×40) field. These foci appeared to be single infected cells in general.
Cell viability analysis
To determine the MDCK cell viability following essential oil treatment, cells were seeded in 24-well tissue culture plates at the density of 5 × 104 cells/well in 500 μl growth medium for adherence. Aliquots of 500 μl varying dilutions from 1:3,000 to 1:18,000 (v/v) of the oil blend in cell growth media were added to each well in triplicate. Cell viability was determined at 7 h and 24 h following protective blend oil exposure using a Trypan blue (0.4% w/v) exclusion assay. Cell viability was counted using a hemocytometer, and expressed as the percentage of trypan blue positive cells over the total number (stained + unstained) of cells.
Hemagglutinin titration of influenza virus
For HA assay, viruses were serially 2-fold diluted in a 96-well plate with CaMg-PBS and an equal volume of 0.5% human red blood cells were added. The plates were kept at 4°C for 60 minutes and agglutination was determined visually.
Flow cytometry analysis and confocal microscopy of IAV binding and internalization
To prepare the cells for flow cytometry and confocal microscopy to examine viral binding and internalization, MDCK cells in exponential growth phase were trypsinized and resuspended in DMEM. Cells (5 × 105) in 100 μl were exposed to PR8 at an MOI of 50, and incubated at 4°C for 30 minutes to allow virus binding. A negative control was performed by exposing cells to an equal volume of sterile virus-free buffer. For oil-treated virus, PR8 was incubated in the protective essential oil at a dilution of 1 to 4,000 for 2 h prior to the exposure. For virus internalization, cells were further incubated with virus at 37°C for 30 minutes. Surface associated virus was removed by incubating the cells in sialidase from Clostridium perfringens (Sigma, St Louis, MO) at 40 mU in 200 μl of CMS/Ac, pH 5.5 (150 mM NaCl, 10 mM CaCl2 and 0.5 mM MaCl2) for 1 h at 37°C with gentle rocking. Following the incubation with virus and/or sialidase, the cells were washed in triplicate to remove unbound virus and then incubated for 20 minutes in 100 μl stain buffer (BD Biosciences, San Jose, CA) with 5% FCS containing 1 μl canine IgG solution as an FcR blocker. After being treated with Perm/Wash Buffer (BD Biosciences) for 10 minutes, the cells were stained with an anti-IAV NP monoclonal antibody followed by incubation with an Alexa Fluor 488-conjugated goat anti-mouse IgG for 40 minutes. The cells were fixed by incubating on ice with 250 μl Cytofix (BD Biosciences) and then washed in triplicate. For FACS analysis, the cells were resuspended in 250 μl Stain Buffer; and single cell suspensions were prepared using a mesh filter. A FACScan cytometer, BD Biosciences LSR II, was used to assess PR8 binding and internalization. A minimum of twenty thousand events were counted for each sample. Analytical gates were set so that ≤1% of negative control cells exceeded the gate. The percentage of cells exceeding the gate was used to determine virus binding and internalization. For confocal microscopy, the cells were resuspended in 200 μl of Stain Buffer and mounted on microscope slides using acrylic-based mounting medium containing DAPI (Invitrogen).
Measurement of viral mRNA expression by relative end-point RT-PCR
MDCK cells were exposed to oil-treated or untreated PR8 at an MOI of 32. For mock infection, cells were exposed to sterile virus-free buffer. After 18 h, total RNA from MDCK cells was extracted using TRIzol (Invitrogen, Carlsbad, CA). The quantity and quality of the isolated RNA was determined spectrophometrically and by formaldehyde agarose gel electrophoresis, respectively. An equal amount (1 μg) of the total RNA was reverse transcribed for first-strand cDNA synthesis. Then an aliquot of the cDNA was subjected to 30 cycles of PCR amplification (95°C, 30 sec.; 55°C, 1 min.; 72°C, 1 min.) in the presence of primer pairs targeting NP, NS1 or GAPDH. The following primer sequences were used: NP sense 5'-AGGACAAGAGCTCTTGTTCG-3', and anti-sense 5'-CTCTTGTGTGCTGGATTCTC-3'; NS1 sense 5'-GACCAAGAACTGAGTGATGC-3', and anti-sense 5'-TGACCTAGCTGTTCTCGCC-3'; GAPDH sense 5'-TGAAGGTCGGAGTCAACGGATTTGGT-3', and anti-sense 5'-CATGTGGGCCATGAGGTCCACCAC-3'. Aliquots of 25 μl PCR products were electrophoresed on 1% agarose gels and visualized by ethidium bromide staining. DNA bands were imaged and quantified using ImageQuant 5.0 software (GE Healthcare, Piscataway, NJ). Levels of NP and NS1 were normalized to the corresponding GAPDH levels for each sample.
Viral NS1 protein determination by immunoblotting
MDCK cells were exposed to oil-treated or untreated PR8 at an MOI of 32. For mock infection, cells were exposed to an equal volume of virus-free buffer. Cells were harvested at 18 h following infection by lysing the cells with RIPA buffer (150 mM NaCl; 50 mM Tris, pH 8.0; 10 mM EDTA, NaF, and sodium pyrophosphate; 1% NP-40; 0.5% sodium deoxycholate; 0.1% SDS; 10 μg of leupeptin/ml). Cell homogenates were clarified by centrifugation at 4°C; and 20 to 30 μg of the lysates were separated on 4-15% gradient SDS-PAGE gels. Proteins were then transferred onto nitrocellulose membranes. Following blocking with 3% non fat milk plus 1% BSA, the membranes were incubated anti-IAV NS1 or anti-β-actin (Abcam) antibody overnight followed by incubation with horseradish peroxidase-conjugated rabbit anti-goat IgG (Cell Signaling Technology). Target protein binding was detected using a chemilluminescent reagent (Pierce Biotechnology, Rockford, IL), and visualized by the Syngene G:box Bioimaging System and GeneTools software (Syngene, Frederick, MD).
Results are reported as the mean ± standard error of mean (SEM) of at least three replicate experiments. All statistical analysis was carried out with GraphPad Instat 3. Statistical significance was determined by one-way ANOVA with Student-Newman-Keuls post hoc correction for multiple comparisons. Statistical significant was considered when p < 0.05.
Protective essential oil suppressed progeny virus production
The effect of oil treatment on viral infectivity was first determined by measuring the release of nascent viral particles following infection of MDCK cells with untreated, oil treated, or control oil treated virus for 48 h.
Virus treated with a 1:4,000 dilution of protective essential oil decreased the infectious particle number by 90% (Figure 1, bottom panel). This did not appear to be due to a toxic effect of the oil on MDCK cells. Because addition of treated virus to MDCK cells resulted in a further dilution of the oil in the media, cells were actually exposed to protective oil at a concentration 1,500 times lower than that used to treat the virus. For example, treatment of the virus with a 1 to 3,000 dilution of oil resulted in exposure of the MDCK cells to a cellular exposure to a 1 to 4,500,000 dilution of oil. This is far below the minimal concentration that caused any detectable cellular cytotoxicity (1: 3,000; Figure 5). Thus, it appeared that the protective oil inhibited IAV PR8 viral production in MDCK cells was not due to non-specific cytotoxicity.
Protective essential oil suppresses virus infection
Effect of oil treatment on HA activity
HA titration comparison of essential oil-treated to untreated influenza virus*.
Essential Oil Dilution
Time (h) after oil treatment
Binding and internalization of virus in MDCK cells
Effect of essential oil on cell viability
To determine if essential oil inhibited virus infectivity and progeny production by direct cellular cytotoxic effects, the viability of MDCK cells was measured after incubation with media in the presence or absence of oil. Viability was determined by morphological examination and trypan blue exclusion. Dilutions of oil corresponding to final concentrations of the cells were also used. This was to account for the fact that after oil exposure of the virus, addition of treated virus to MDCK cells resulted in a further dilution of the oil in the media. Therefore, dilutions of protective oil (1:3,000 to 1:18,000) in cell growth media, corresponding to working dilutions for cells in FFA assay, were added to each well in triplicate. For these experiments, two incubation times were used. Seven-hour incubation was used to duplicate the exposure of MDCK cells to oil during the assay for the effect of oil on the first cycle of virus infection (see Figure 2). Twenty-four hours of exposure were used to duplicate the oil exposure during the assay for the effect of oil treatment on viral progeny production (see Figure 1).
Morphologically, oil-treated cells did not show signs of death at 7 h. At 24 h of exposure, MDCK cells remained attached to the bottom of plates and did not show noticeable morphological alterations at the dilution up to 1:3,000.
MDCK cells infected by oil-treated virus express viral mRNA, but minimal amounts of protein
In order to develop novel therapies, many plant extracts have been tested for antiviral activity. Over the last decade, laboratory research has found that many essential oils diminish viral infectivity. For example, Melissa officinalis oil decreases infectivity of enveloped herpesviruses . Essential oils from hyssop, thyme or ginger have inhibitory effects on herpes simplex virus type 2 (HSV-2) in vitro as determined by plaque assay in RC-37 cells .
With regard to IAV, Melaleuca alternifolia essential oil has antiviral activity against influenza PR8 virus and that antiviral activity has been principally attributed to a specific component, terpinen-4-ol . The anti-IAV activity is manifested by inhibition of virus-induced cytopathogenicity. Also, a polyphenol rich extract (CYSTUS052) from the Mediterranean plant Cistus incanus exerts potent anti-influenza virus activity in A549 or MDCK cell cultures infected with IAV. On a molecular basis the protective effect of CYSTUS052 appears to be mainly due to binding of the polymeric polyphenol components of the extract to the virus surface, thereby inhibiting binding of the hemagglutinin to cellular receptors . Echinacea purpurea extract has activity against highly pathogenic avian influenza virus, via inhibition of cell receptor binding activity of the virus .
Replication of the virus RNA genome in infected cells was found to be constantly suppressed in the presence of the Agrimonia pilosa extract. The data suggests that the extract exerts an antiviral effect on multiple rounds of the influenza infection cycle, primarily the initial step of the virus life cycle, but activity at later stages, including replication and transcription, cannot be ruled out . Study of catechins in green tea on influenza virus suggests that the antiviral effect of catechins on influenza virus is mediated not only by specific interaction with HA, but altering the physical properties of viral membrane . Here, we report that a blend of essential oil attenuates IAV infection in MDCK cells. Our work shows that the mechanism of antiviral activity is due to inhibition of viral protein synthesis. This mechanism of inhibition is similar to that of trans-cinnamaldehyde (CA), one of the principal constituents of essential oil derived from Cinnamomi cortex. This extract inhibits the growth of influenza PR8 virus in vitro and inhibited viral protein, but not mRNA synthesis . In this study, they were also able to demonstrate a protective effect of CA in PR8 infected mice. Thus, the inhibitory activity of plant extracts appears to be at multiple stages of viral infection and proliferation.
Some traditional herbal medicines have been used for the treatment of various diseases for more than 2,000 years. Recently some have been officially approved for clinical use. For example, aspirin was originally made from powder of the bark and leaves of the willow tree to help heal headaches, pains and fevers back in Hippocrates days. Currently, 148 Japanese kampo medicines have now formally been approved by the Ministry of Health, Labor and Welfare of Japan for use in clinics. In addition, several kampo were shown to have immuno-modulatory and anti-viral effects both in vitro and in vivo. With more understanding of their antiviral mechanisms, more traditional medicines will be utilized for clinical pharmaceutical purposes and novel drug discovery.
The lack of toxicity and potent specific viral inhibitory activity suggest essential oil may be helpful as a possible antiviral drug for control and treatment of influenza virus infection. It could potentially be used as a non-toxic way to cleanse surfaces, or dispersed to eliminate aerosolized virus particles in confined areas. Since the oil is currently used as a food supplement, oral administration, once the pharmacokinetics are determined, may provide therapeutic benefit during infection.
We have shown that virus treated with protective essential oil significantly decreased both the number of released viral particles from infected MDCK cells, and infected cells by FFA assay. Also, oil treated virus had the same ability to bind to and internalize in MDCK cells compared with untreated virus. MDCK cells infected by oil-treated virus express viral mRNA, but minimal amounts of protein. Taken together, we found an essential oil blend notably attenuates influenza virus PR8 infection in vitro via inhibition of viral protein synthesis at the post-transcription level. The lack of toxicity and potent specific inhibition ability make the essential oil a possible antiviral drug for influenza virus proliferation control and treatment.
The research described in this work was partially supported by a Clinical Innovator Award from the Flight Attendant Medical Research Institute and by the Oklahoma Health Research Program from Oklahoma Center for the Advancement of Science & Technology (to W. W.).
We thank Elizabeth Duggan for excellent technical support. We also acknowledge the assistance from the Oklahoma Medical Research Foundation Imaging Analysis core facility.
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