Protective essential oil attenuates influenza virus infection: an in vitro study in MDCK cells

Shuhua Wu, Krupa B Patel, Leland J Booth, Jordan P Metcalf, Hsueh-Kung Lin, Wenxin Wu, Shuhua Wu, Krupa B Patel, Leland J Booth, Jordan P Metcalf, Hsueh-Kung Lin, Wenxin Wu

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Figures

Figure 1
Figure 1
Effect of oil treatment on progeny virus production by PR8 as measured by Fluorescent focus assay (FFA). After MDCK cells in 24-well plates were infected with oil-treated and untreated virus for 48 h, five microliters of supernatants were removed, serially diluted and added to confluent MDCK cells in 96-well plates. After incubation for 7 h, IAV nucleoprotein (NP) was detected using an Alexa Fluor 488 (green) labeled antibody. Panels: (A) MDCK cells unexposed to virus, but stained with anti-NP antibody. Panels (B-F) MDCK cells exposed to PR8 treated with different dilutions of essential oil: (B) 1:1,000 (C) 1:2,000 (D) 1:3,000 (E) 1:4,000 (F) 1:6,000, (G) untreated PR8, (H) PR8 treated with control oil at a 1:1,000 dilution. Panels I-L were fluorescence images merged with corresponding brightfield images to show MDCK cell morphology: (I) PR8 treated with essential oil 1:4,000, (J) PR8 treated with essential oil 1:6,000, (K) untreated PR8, (L) PR8 treated with control oil 1:1,000 (merge). Bottom panel: Infectivity as reflected by the percentage of cells in which IAV NP was detected. The results represent the mean ± SEM from three independent experiments.
Figure 2
Figure 2
Effect of essential oil on the first cycle of PR8 infection as determined by FFA. MDCK cells in 96-well plates were infected by oil-treated virus for 7 h, and viral NP was detected using an Alexa Fluor 488 (green) labeled antibody. Panels: (A) MDCK cells unexposed to virus, but stained with anti-NP antibody. Panels B-F cells exposed to PR8 treated with dilutions of essential oil (B) 1:1,000 (C) 1:2,000 (D) 1:3,000 (E) 1:4,000 (F) 1:5,000, (G) 1:6,000, (H) untreated PR8, (I) PR8 treated with control oil 1:1,000. Panels I-L were fluorescence images merged with corresponding brightfield images to show MDCK cell morphology (J) PR8 treated with essential oil 1:1,000, (K) PR8 treated with essential oil 1:6,000, (L) untreated PR8 (merge). Bottom panel: PR8 first cycle NP production as depicted by the percentage of cells containing detectable NP at 7 h after infection. The results represent the mean ± SEM from three independent experiments.
Figure 3
Figure 3
Essential oil dose not block IAV PR8 binding and entry to MDCK cells as determined by flow cytometry. MDCK cells were exposed to oil-treated (1:4,000 dilution) and untreated PR8. Viruses were allowed to bind to the cells at 4°C for 30 minutes, or allowed to bind and then internalize at 37°C for 30 minutes. Following the incubation of selected samples with sialidase treatment, viral NP was stained with the fluorescent dye Alexa Fluor 488. The percentage of cells exceeding the analytical gate was used to determine viral binding and internalization. The data are representative of three separate experiments.
Figure 4
Figure 4
Essential oil dose not block IAV PR8 binding and entry in MDCK cells as confirmed by confocal imaging. Binding and internalization of oil treated (1:4,000 dilution) and untreated PR8 virus to MDCK cells was examined as described in Figure 3. Following infection, NP was stained with the fluorescent dye Alexa Fluor 488. Cell nuclei were stained with DAPI (purple).
Figure 5
Figure 5
Effects of essential oil treatment on PR8 production are not due to cytotoxicity. Cell viability was determined using trypan blue exclusion at 7 and 24 h of essential oil exposure. Data were presented as the mean ± SEM from at least 3 independent experiments. Means were compared to data from the control oil group. **P < 0.01.
Figure 6
Figure 6
Essential oil treatment does not inhibit PR8 NP mRNA expression in MDCK cells. After infection of MDCK cells with oil-treated (1:4,000 dilution) and untreated PR8 virus at 4°C or 37°C for 18 h, total RNA was extracted and PR8 NP mRNA expression was assessed by relative end-point RT-PCR (A). Transcript levels of NP normalized relative to the constitutively expressed GAPDH gene (B). The data are representative of three separate experiments.
Figure 7
Figure 7
MDCK cells infected by oil-treated virus express viral NS1 mRNA, but minimal amounts of NS1 protein. After infection of MDCK cells with oil-treated (1:4,000 dilution) and untreated PR8 virus at 4°C and 37°C for 18 h. Total RNA was extracted from cells and PR8 NS1 mRNA expression was assessed by relative end-point RT-PCR (A). Transcript levels of NS1 normalized relative to the constitutively expressed GAPDH gene (B). Western blot was used to determine NS1 protein expression in the infected cells (C). The membranes were probed with anti-NS1 or anti-actin antibodies. The data depicted are representative of three separate experiments.

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Source: PubMed

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