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.

Quantification of infectious particles produced in virus-exposed MDCK supernatant was done by transfer of the infected cell supernatant to a separate culture of MDCK cells followed by Fluorescent focus assay (FFA). The fluorescent foci were counted by fluorescent microscopy. There was no detectable green fluorescent signal in cells exposed to virus-free diluents (Figure 1A, top panels). Oil treatment inhibited nascent PR8 production and release into infected supernatants in a dose dependent manner (Figure 1). Virus treatment with control canola oil (1:1,000) had no significant effect on viral particle production (Figure 1G and 1H, top panels). IAV pretreatment with protective oil decreased production of viral particles (Figure 1B-F, top panels).

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. Full size image

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

To determine whether protective oil directly inhibited the first cycle of IAV infection, oil-treated PR8 was added to MDCK cells and direct FFA assay of the infected cells was performed. Fluorescent foci of infected cells were easily detectable after 7 h of exposure to untreated or control canola oil (1:1,000) treated PR8. The nascent produced nucleoprotein (NP) by FFA was significantly decreased by virus treatment by oil at concentrations greater than 1 to 3,000 (Figure 2B-D, top panels). Treatment of PR8 with a 1 to 3,000 dilution of oil decreased the number of infected MDCK cells by 50% (Figure 2, bottom panel). As the oil was further diluted, the final oil dilutions applied to MDCK cells during exposure to virus were 3 fold higher (more dilute) than those used to treat virus in this experiment. For example, treatment of virus with a 1 to 3,000 dilution of oil resulted in MDCK cell exposure of 1 to 9,000. Thus, the protective essential oil significantly inhibited IAV PR8 viral protein production in MDCK cells at concentrations that did not appear to be directly toxic to cells.

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. Full size image

Effect of oil treatment on HA activity

To determine whether the effect of protective oil on virus infectivity was due to alterations in virus particle integrity, the effect of oil treatment on HA activity was assessed. HA activity was measured in untreated PR8 virus or virus treated with various dilutions of protective oil or control oil for 24 h, 48 h and 72 h. Although HA activity decreased with time for all treated and mock treated viruses, there was no significant effect of oil treatment on HA activity (Table 1) at several concentrations that decrease viral infectivity and progeny virus production. There was a modest effect of a 1 to 1,000 dilution on HA activity after 72 h oil treatment. This effect was not seen when dilutions greater than 1:1,000 were used, even with 72 h of exposure. This data shows that the effect of oil treatment on infectivity and viral progeny production was not due to inhibition of HA activity.

Table 1 HA titration comparison of essential oil-treated to untreated influenza virus*. Full size table

Binding and internalization of virus in MDCK cells

In order to examine the mechanism of inhibition of PR8 by oil treatment, the effect of this treatment on virus internalization was studied. To this end, we developed a flow cytometry (FACS) internalization assay. Virus was detected using an Alexa Fluor 488 labeled secondary antibody against monoclonal antibody to PR8 NP. At 4°C, virions attach to the host cell surface, but internalization does not occur. Sialidase is then used to remove the bound, but not internalized virus. Cell associated virus present after sialidase treatment is presumed to be internalized, and this was indeed confirmed by confocal microscopy (see below). FACS assay demonstrated as expected that PR8 bound to receptors at the cell surface but was not internalized at 4°C as the cell-associated fluorescence of stained influenza virus was removed by sialidase (Figure 3, left panels). At 37°C, PR8 internalization occurred, as cell-associated fluorescence was not removed by sialidase treatment (Figure 3, left panels). As determined by the FACS assay, oil treatment did not affect virus binding to MDCK cells. Also, internalization was not detectably affected by oil treatment of PR8 as determined by the amount of sialidase-resistant cell associated fluorescence after incubation of virus exposed cells at 37°C (Figure 3E, L, right panels).

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. Full size image

Results from confocal microscopy confirmed the results of the flow cytometry assay for virus binding and internalization (Figure 4). At 4°C, viruses remained on the cell surface as shown as a green fluorescent ring around the cell membrane (Figure 4, panel A and F). In contrast, the viruses were present in the cytoplasm after incubation at 37°C (Figure 4, panel C and H). Cell-associated influenza virus in cells incubated at 4°C was removed by sialidase, but not after incubation at 37°C confirming that sialidase treatment removed the surface bound, but not internalized, virus (Figure 4, panel B, G, D and I). There was no apparent effect of oil treatment on binding and internalization of PR8. Together, these data demonstrate that oil treatment does not appear to inhibit IAV infectivity and progeny production by alteration of virus binding and internalization.

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). Full size image

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.

Cell viability by trypan blue exclusion was also determined at 7 and 24 h following exposure to the essential oil blend. At all concentrations of oil, all MDCK cells were alive after 7 h of incubation. After 24 h, cell viability was not significantly affected by the increasing concentrations of protective blend oil up to a 1: 3,000 dilution (Figure 5). At this concentration about 60% of the cells were dead. Control oil (canola oil, 1:1,000) did not cause any cell death after 24 h. As there was no effect of any concentration the oil on cell viability at 7 h the effect of oil treatment on the first cycle of viral infection does not appear to be due to cytotoxicity. Also, as the oil concentrations causing cytoxicity after 24 h of incubation were much greater than those that inhibited viral progeny production, it appears that this effect of oil treatment is not due to cytotoxic effects of the oil.

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. Full size image

MDCK cells infected by oil-treated virus express viral mRNA, but minimal amounts of protein

We next sought to determine whether the effects of oil treatment on viral infectivity and progeny production could be due to inhibition of viral gene expression at the transcriptional level. Endogenous mRNA levels of viral NP were determined using relative end-point RT-PCR. As expected, when cells were exposed to virus at 4°C, no viral NP RNA was detected, consistent with virus binding, but failing to internalize under these conditions. At 37°C, RNA expression of NP was detected in cells infected with both oil-treated virus and untreated PR8 virus. NP mRNA expression levels were similar whether oil-treated or untreated virus was used (Figure 6). This finding suggests that the decrease in NP protein expression seen with oil treatment (see Figure 2) was likely due to inhibition of viral mRNA transcription.

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. Full size image

To confirm whether oil treatment inhibited viral protein but not mRNA production, we also measured mRNA and protein expression levels of another viral protein, NS1, in control and oil treated virus-exposed cells. As with viral NP, mRNA expression of NS1 was not affected by oil treatment of PR8 (Figure 7A). In contrast, NS1 protein expression was significantly decreased by treatment of PR8 with essential oil. (Figure 7B). Based on the above results, inhibition of viral progeny production and infection by essential oil is likely due to inhibition of viral protein synthesis.