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 . 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 [, 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 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.
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 1st 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 2nd 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.
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 1st antibody, as described above (2.4). Incubation with 2nd 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).
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.
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).