Anti-viral properties and mode of action of standardized Echinacea purpurea extract against highly pathogenic avian Influenza virus (H5N1, H7N7) and swine-origin H1N1 (S-OIV)

Echinaforce^® (EF) and Virus Concentration

We reported previously that at concentrations up to 1.6 mg/ml (dry mass/vol, the recommended oral dose) the EF extract showed no apparent cytotoxic effects, according to trypan blue staining, MTT assays, or microscopic examination [[21], data not shown]. However at concentrations of >1.6 μg/ml ≥99% inactivation of H3N2-type IV was achieved (Table 1). The degree of inactivation depended on the virus dose, as might be expected (Fig. 1). MIC₁₀₀ values increased from 0.32 μg/ml for 10² PFU/ml virus, up to 7.5 μg/ml for 10⁵ PFU/ml.

In order to exclude the possibility that the virucidal effect might be subtype specific or related only to human IV, we analyzed the effect of EF in non toxic concentrations on a human isolate of a H5N1-type HPAIV (KAN-1). Virus yield reduction assays were carried out with KAN-1, which had been pre-incubated with various concentrations of EF, from 0.1 to 50 μg/ml (Fig. 2). At the highest concentration the yield was reduced by more than 3 log₁₀. Furthermore we tested the inhibitory effect of EF on human H1N1-type (PR8) and a H7-type HPAIV (FPV) and obtained comparable results (data not shown), indicating that EF affects not only human IV (H3N2, H1N1) but also both types (H5, H7) of HPAIV (data not shown).

Time of addition of Echinaforce^®

All the IV strains tested, the human pathogenic Victoria (H3N2), PR8 (H1N1), S-OIV (H1N1), and the avian strains KAN-1 (H5N1) and FPV (H7N7) were susceptible to the EF, but only as a result of direct contact. Pre-incubation of cells with extract, followed by virus infection, or post-exposure of the infected cells to EF, inhibited virus replication to a lesser extent (data not shown). To investigate this in more detail, several experiments were performed with KAN-1 to determine the effect of adding EF at different times relative to virus infection of the cells. Complete inhibition was achieved by incubating KAN-1 and EF together before adding to the cells (Fig 3, lanes 3, 4, and 6). However other combinations of pre- and post-exposure to EF (lanes 2, 5, and 7) resulted in only partial reduction in virus production, compared to untreated (lane 1). These results suggest that EF was acting either directly on the virus or at a very early stage in the replication cycle. It is noteworthy to mention, that removal of EF containing medium 6.5 hours p.i. and further incubation in normal medium for 1.5 hours in order to prevent an exposure of newly formed virions to EF prior to titration, did not change this result.

Intra-cellular RNP localization

Next, the production and intra-cellular localization of viral RNP were determined by immunofluorescence, in MDCK cells infected with KAN-1, with and without EF treatment (Fig 4). In normally infected cells (- EF), the nucleocapsid protein (NP, green), which is the main component of the RNPs, appeared initially in the nucleus (6 hours) followed by migration to the cytoplasm (8 hours). The same pattern was seen in EF-treated cells infected with untreated virus (cells + EF), and in cells exposed to EF after infection (EF p.i.). However, when cells were infected with EF-treated virus (virus + EF), the overall number of positive cells was significantly reduced. Nevertheless, the amount and the localization of RNPs detected in cells infected with pre-treated IV was the same as for untreated cells infected with untreated virus. It should be noted that the treatment of infected cells at different time points p.i. did not affect the number of cells positive for NP staining (data not shown). These results suggest that EF affects a very early stage before replication, but once the virus has entered the cells its replication and spread are not affected.

Interaction of Echinaforce^® with Viral HA

Lack of Resistance to Echinaforce^®

Treatment with currently available anti-influenza drugs directly targeting the virus has the drawback that, due to the high mutation rate of IV, resistant strains will inevitably arise. This has been shown for neuraminidase inhibitors like Tamiflu^® in regard to seasonal IV, H5N1 HPAIV and in recent reports for the pandemic S-OIV [2, 3, 9, 27, 28]. Therefore, any competitive alternative should have the advantage of preventing emergence of resistant IV variants [11]. This might be different for a substance that unspecifically blocks virus activity. The possibility of emergence of EF-resistant virus was evaluated by comparing relative H5N1 virus yields in the presence and absence of EF, or Tamiflu^®, during consecutive passages through cell cultures. Results are shown in Fig 5. After one round of replication virus yields were substantially reduced by 50 μg/ml EF or 2 μM Tamiflu^®. However in rounds 2 and 3 the yields in the presence of Tamiflu^® were similar to controls, indicative of emergence of resistant virus variants, whereas in the presence of EF yields continually remained low, indicating lack of EF-resistant virus.

To determine if Tamiflu^®-resistant virus remained sensitive to EF, the growth of Tamiflu^®-resistant virus (produced in the above experiments) was tested in the presence and absence of EF. EF (50 μg/ml) reduced the yield of Tamiflu^®-resistant virus by more than 3 log₁₀ viral FFU, similar to that of standard virus (data not shown).

Standard Echinacea Preparation

Echinaforce^® (obtained from A. Vogel Bioforce AG, Roggwil, Switzerland) is a standardized preparation derived by ethanol extraction of freshly harvested Echinacea purpurea herb and roots (95:5). The composition of marker compounds (ie. those compounds known to characterize this species of Echinacea) was described previously [21]. The concentration of ethanol was 65% v/v. The final concentration of ethanol in the experimental reactions and cultures was too low to cause adverse effects on the cells or viruses. In addition the preparation was free of detectable endotoxin (as determined by means of a commercial assay kit, Lonza Walkersville Inc., MD, lower limit of detection 0.1 unit/ml), and the administered amount that was effective in our experiments, up to the recommended oral dose of 1.6 mg/ml, was not cytotoxic according to trypan blue staining, MTT formazan assays (MTT = 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan), and microscopic examination [[21], and data not shown].

Cell lines & Viruses

Madin-Darby canine kidney cells (MDCK) were acquired originally from ATCC and were passaged in Dulbecco's MEM (DMEM), in cell culture flasks, supplemented with 5-10% fetal bovine serum, at 37°C in a 5% CO₂ atmosphere (cell culture reagents were obtained from Invitrogen, Ontario (CA) or Karlsruhe (DE)). No antibiotics or anti-mycotic agents were used for experiments performed in the Hudson laboratory. In the Pleschka laboratory the cell cultures were also grown in DMEM, 10% FCS but supplemented by 100 U/ml penicillin and 100 μg/ml streptomycin (P/S).

The following influenza A virus strains were used: human strain A/Victoria/3/75 (Victoria, H3N2) acquired from the BC Centre for Disease Control, Vancouver. The human HPAIV isolate A/Thailand/KAN-1/2004 (KAN-1, H5N1) was provided to S. Pleschka by P. Puthavathana, Thailand; the HPAIV A/FPV/Bratislava/79 (FPV, H7N7) and the human strain A/Puerto Rico/8/34 (PR8, H1N1) were obtained from the IV strain collection in Giessen, Germany; the human isolate of the 2009 pandemic IV of swine-origin A/Hamburg/1/09 (S-OIV, H1N1) was provided to S. Pleschka by M. Matrosovich, Marburg, Germany. KAN-1 and FPV or PR8 were propagated on MDCK cells with low serum but without trypsin (KAN-1, FPV) or in embryonated chicken eggs (PR8), respectively. All other strains were propagated on MDCK cells in the presence of trypsin (2.5 μg/ml). Stock viruses were prepared as clarified cell-free supernatants or allantois fluid, respectively, with titers ranging from 10⁷ to 10⁸ PFU (plaque-forming units) per ml. and stored at -75°C. Strains were titrated either by standard plaque assay or by focus forming assays (see below).

MIC₁₀₀ values

MIC₁₀₀ values of EF were determined from CPE-endpoint assays, as follows: The Echinacea extract, in 200 μl aliquots, was serially diluted two-fold across replicate rows of a 96-well tray, in medium, starting at the recommended oral dose of 1.6 mg/ml. Virus, 100 PFU in 100 μl, was added to each well and allowed to interact with the extract for 60 min at 22°C. Following the incubation period, the mixtures were transferred to another tray of cells from which the medium had been aspirated. These trays were then incubated at 37°C, 5% CO₂ until viral CPE were complete in control wells containing untreated virus (usually 2 days). Additional wells contained cells not exposed to virus. The MIC₁₀₀ was the maximum dilution at which CPE was completely inhibited by the extract. In most assays the replicate rows gave identical end-points; when two-fold differences were encountered arithmetic means and standard deviations were calculated. In the alternative (intra-cellular) method, the cells were incubated with the diluted extracts first, before adding virus.

Virus titrations

Strain Victoria (H3N2) was titrated by standard plaque assay techniques in MDCK cells with agarose overlays. The other strains were assayed by focus formation in MDCK cells as follows: Cells were grown overnight (to 90% confluency) in complete medium in 96-well trays, washed and inoculated with 50 μl of serially diluted (10^-1 to 10^-8) virus in PBS containing 0.2% BA, 1 mM MgCl₂, 0.9 mM CaCl₂, 100 U/ml penicillin and 0.1 mg/ml streptomycin (PBS/BA), for 60 min at room temperature. The inoculum was replaced by 150 μl MC media (1× DMEM, BA, P/S, 1.5% methyl cellulose). Cells were incubated at 37°C, 5% CO₂ for 44 hours. To detect foci of infection the cells were permeabilized with 330 μl fixing solution (4% paraformaldehyde, 1% triton X-100, in PBS) and stored at 4°C for 60 min followed by 3 washes with PBS/0.05% Tween 20, and incubation with 50 μl 1^st antibody (mouse anti-influenza A nucleoprotein mAb, BIOZOL BZL 10908) diluted in PBS/3% BA at room temperature for 60 min. Cells were then washed 3 × with PBS/Tween 20 and incubated with 2^nd antibody (anti-mouse HRP antibody Santa Cruz sc2005) diluted in PBS/3% BA at room temperature for 60 min. Finally cells were washed 3 × with PBS/Tween20 and incubated in 40 μl AEC staining solution (3-amino-9-ethylcarbazole, Sigma Chemical, AEC #101) for 60 min followed by washing in dH₂O. Foci were scanned and analyzed by means of Photoshop software (Adobe). All titrations were performed in duplicate.

Pre-incubations

In some experiments aliquots of virus (H3N2 or H5N1) in PBS/BA or the cells in complete medium, were pre-incubated with EF (50 μg/ml) at room temperature or 37°C respectively for 60 min, prior to infection. Infected cells and controls were then incubated in medium containing EF (50 μg/ml) at 37°C, 5% CO₂ for 24 hours, at which time supernatants were removed for focus assays.

Intra-cellular RNP localization

Cells were grown and infected on cover slips, and pre-incubations of the viruses were carried out as described above. Cells were fixed, at different times post infection (p.i.), washed with PBS and incubated with 1^st antibody, as described above (2.4). Incubation with 2^nd antibody (rabbit anti-mouse Texas red) diluted in PBS/3%BA was carried out at room temperature for 60 min in the dark. Cells were washed again and incubated with DAPI (0.1 mg/ml PBS/3%BA, Roth Germany) for 10 min in the dark to stain nuclei. After further washing the cover slips with cells were covered with Moviol + DABCO (Moviol, Aldrich, glycerine, Merck, ddH2O, Tris-Cl pH 8.5 + 1,4-Diazobicyclo [2.2.2]octane, Merck) on glass slides. Cells were examined and digitized with a TCS SP5 confocal laser scanning microscope (Leica, Germany).

Hemagglutination assay

25 μl EF in PBS at the indicated concentrations were added to wells of a 96-well tray. Thereafter 25 μl of virus with ca. 2560 HAU/ml were added. The plates were incubated for 60 min at 4°C. After this incubation period, 50 μl of chicken erythrocyte suspension (CES, 0.5% in PBS) were added to each well. The plates were further incubated for 60 min or 4 hours at 4°C. Wells were visually inspected for the presence or absence of hemagglutination. Positive and negative controls without EF treatment or without virus were included. To assay possible hemagglutination by EF itself, 50 μl of EF in PBS at the indicated concentrations were incubated with 50 μl CES for 60 min or 4 hours at 4°C. All assays were performed in quadruplicate.

Virus Resistance Assay

MDCK cells grown over night at 37°C and 5% CO₂ were pre-incubated with 2 ml complete medium (1× DMEM, 10% FCS, Pen/Strep) with or without EF (50 μg/ml), at 37°C and 5% CO₂ for 60 min. In parallel virus in PBS/BA was incubated with EF (50 μg/ml) or left untreated for 60 min. After the pre-incubation period the cells were washed and infected with 500 μl virus suspension (MOI = 0,001) (+/-) Echinaforce^® (50 μg/ml). Cells were then incubated for 60 min in the dark at room temperature after which the inoculum was removed. Cells were further incubated in 2 ml medium (DMEM/BA/P/S with Echinaforce^® (50 μg/ml), Tamiflu^® (2 μM) or without test substances) at 37°C, 5% CO₂ for 24 hours. Samples of the supernatants were collected, which were then assayed by focus forming assay for further determination of infectious virus. Following the assays, these supernatants were used to infect another set of cultures under the same conditions as described above. This process of sequential infection with supernatants was repeated once more to yield in total three rounds of infection and replication. Experiments done in duplicates were stopped when the Tamiflu^® sample reached titers of the untreated control.

Biosafety

All experiments with infectious virus were performed according to German and Canadian regulations for the propagation of influenza A viruses. All experiments involving highly pathogenic influenza A viruses and the pandemic S-OIV were performed in a biosafety level 3 (BSL3) containment laboratory approved for such use by the local authorities (RP, Giessen, Germany).