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)

Background Influenza virus (IV) infections are a major threat to human welfare and animal health worldwide. Anti-viral therapy includes vaccines and a few anti-viral drugs. However vaccines are not always available in time, as demonstrated by the emergence of the new 2009 H1N1-type pandemic strain of swine origin (S-OIV) in April 2009, and the acquisition of resistance to neuraminidase inhibitors such as Tamiflu® (oseltamivir) is a potential problem. Therefore the prospects for the control of IV by existing anti-viral drugs are limited. As an alternative approach to the common anti-virals we studied in more detail a commercial standardized extract of the widely used herb Echinacea purpurea (Echinaforce®, EF) in order to elucidate the nature of its anti-IV activity. Results Human H1N1-type IV, highly pathogenic avian IV (HPAIV) of the H5- and H7-types, as well as swine origin IV (S-OIV, H1N1), were all inactivated in cell culture assays by the EF preparation at concentrations ranging from the recommended dose for oral consumption to several orders of magnitude lower. Detailed studies with the H5N1 HPAIV strain indicated that direct contact between EF and virus was required, prior to infection, in order to obtain maximum inhibition in virus replication. Hemagglutination assays showed that the extract inhibited the receptor binding activity of the virus, suggesting that the extract interferes with the viral entry into cells. In sequential passage studies under treatment in cell culture with the H5N1 virus no EF-resistant variants emerged, in contrast to Tamiflu®, which produced resistant viruses upon passaging. Furthermore, the Tamiflu®-resistant virus was just as susceptible to EF as the wild type virus. Conclusion As a result of these investigations, we believe that this standard Echinacea preparation, used at the recommended dose for oral consumption, could be a useful, readily available and affordable addition to existing control options for IV replication and dissemination.


Background
Influenza viruses (IV) continue to cause problems globally in humans and their livestock, particularly poultry and pigs, as a consequence of antigenic drift and shift, resulting frequently and unpredictably in novel mutant and re-assortant strains, some of which acquire the ability to cross species barriers and become pathogenic in their new hosts [1]. Prospects for the emergence of pandemic strains of swine and avian origin have been discussed in several recent reports [2,3]. Some of the highly pathogenic avian IV (HPAIV) strains, in particular H5N1, have occasionally infected humans and pose a severe threat because of their high pathogenicity, with mortality rates exceeding 60% [4,5].
The practicality and efficacy of control by timely vaccination has been questioned [1,6,7], and potential control of IV by synthetic anti-viral chemicals has usually been thwarted by the inevitable emergence of resistant strains, a situation that has been documented in the case of the M2 ion-channel inhibitors, such as adamantane derivatives, and the neuraminidase inhibitors such as oseltamivir and zanamivir [8,9]. Virus-strain specificity is another limitation in the use of these inhibitors.
Alternative approaches to therapy that overcome these obstacles are urgently needed and have been suggested. These include manipulation of specific signaling pathways known to be involved in virus replication [10,11]. As such, the Raf/MEK/ERK-signal transduction cascade and activation of the transcription factor NF-κB were shown to be essential for efficient nuclear export of the viral ribonucleoprotein (RNP) complexes. They have proven to be highly interesting targets, as their inhibition significantly reduces virus replication without emergence of resistant variants in vitro and in vivo [12][13][14][15]. Another approach is the use of broad-spectrum and chemically-standardized anti-IV herbal extracts and compounds with demonstrated efficacy in vitro [16][17][18][19]. These could conceivably afford a more generalized inhibition of all virus strains, either by virtue of inactivating the virus directly or by interfering with one or more essential stages in virus replication or dissemination. Furthermore anti-viral herbal extracts frequently exhibit multiple bioactivities [20], and this could enable their use at relatively low doses of the active compounds, possibly acting in synergy, while at the same time providing a relatively safe "drug" with few side effects. Needless to say, acquisition of resistance to herbal compounds is also a potential problem; consequently this would need to be evaluated, although if multiple bioactive compounds were involved, this would substantially reduce the risk of resistant viruses emerging.
We recently reported the anti-viral properties of a standardized preparation of Echinacea purpurea (Echinaforce ® , EF), which has become a very popular herbal "remedy" for the symptoms of "colds and flu". In addition to possessing potent virucidal activity against several membrane containing viruses, including H3N2-type IV, at the recommended dose for oral consumption, the preparation also effectively reversed virus-induced pro-inflammatory responses in cultured epithelial cells [21]. Some Echinacea-derived preparations also possess selective anti-bacterial and immune modulation activities that might also contribute to their beneficial properties [22,23]. However, our studies also indicated that anti-viral and cytokineinhibitory properties vary widely among different Echinacea species and components [24][25][26]; thus it is important to carry out research on Echinacea preparations that have been standardized and chemically characterized.
The objective of the present study was to investigate the anti-IV activity in more detail, and to elucidate possible mechanisms of action on a variety of IV strains (human and avian), with emphasis on a human isolate of the H5N1-type HPAIV, and to evaluate the potential for emergence of resistant strains, in comparison with oseltamivir (Tamiflu ® ).

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). MIC 100 values increased from 0.32 μg/ml for 10 2 PFU/ml virus, up to 7.5 μg/ml for 10 5 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 log 10 . 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 postexposure 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 replica-tion 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
The first step in entry of IV into cells depends on the interaction between the viral HA and a specific cellular sialic MIC depends on the viral dose Figure 1 MIC depends on the viral dose. Increasing amounts of IV (Victoria, H3N2) were used to determine the MIC 100 of EF. Serial dilutions of EF, in quadruplicate, were incubated with the amounts of IV indicated (10 2 , 10 3 , 10 4 , 10 5 PFU), and transferred to cells for CPE-endpoint determination, as described in Materials and Methods section. The MIC 100 (μg/ ml) is the concentration of EF that leads to complete prevention of CPE.  Table  2. EF inhibited HA activity for all 3 virus strains, in a concentration and time-dependent manner. The same concentrations of EF without virus showed no hemagglutination, as expected (data not shown). In addition there was no visual evidence of erythrocyte lysis in any of the reactions. Therefore the inhibition in HA activ-ity was due to an interference by EF. As this is effective against different human and avian strains, EF might exert an unspecific effect on IV replication by interfering with viral receptor binding and entry.

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 sub-Pre-treatment of IV with EF is most effective Figure 3 Pre-treatment of IV with EF is most effective. H5N1 HPAIV (MOI = 1) and MDCK cells were treated with EF (50 μg/ml) as indicated. Infected cells were then incubated in medium with or without EF for 8 and 24 hours and the infectious titer was determined (FFU/ml). The experiment was performed in triplicate, and titrations in duplicate. 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 log 10 viral FFU, similar to that of standard virus (data not shown).

Discussion
These results have shown that Echinaforce ® (EF), a standardized Echinacea purpurea extract, has potent anti-viral activity against all the IV strains tested, namely human Victoria (H3N2) and PR8 (H1N1), avian strains KAN-1 (H5N1) and FPV (H7N7), and the pandemic S-OIV (H1N1). Concentrations ranging from 1.6 mg/ml, the recommended dose for oral consumption, to as little as 1.6 μg/ml of the extract, a 1:1000 dilution, could inactivate more than 99% of virus infectivity, and treated virus gave rise to markedly reduced yields of virus in cell culture. However, direct contact between virus and EF was required for this inhibitory effect, since pre-treatment of cells before virus infection, or exposure of cells p.i. to EF, led to substantially less inhibition, indicating that the anti-viral effect was manifest at a very early stage in the infection process. This was then confirmed by the use of hemagglutination assays, which clearly showed that EF inhibited HA activity and consequently would block entry of treated virus into the cells. Nevertheless, the mechanism of this inhibition needs to be studied in more detail.
The general inhibition of EF against the different virus strains constitutes a significant advantage over other strain specific anti-virals, such as adamantanes [8,9]. Furthermore, the lack of emergence of EF-resistant viruses during sequential passage is a significant advantage over Tamiflu ® , which under similar culture conditions readily allowed resistant virus strains to develop. In addition the Tamiflu ® -resistant virus was still very sensitive to EF. These results indicate that EF could be helpful in IV control, and would be complemented by the known ability of EF to counteract pro-inflammatory cytokine and chemokine induction caused by IV and other viruses, as well as the selective anti-bacterial activities of Echinacea extracts [23]. Thus EF could play a multi-functional role during IV infections.
Intra-cellular RNP production and localization is not affected by EF Figure 4 Intra-cellular RNP production and localization is not affected by EF. H5N1 HPAIV (MOI = 1) and MDCK cells were either left untreated or were treated with EF as follows: (   The Echinaforce ® extract contains known concentrations of potentially bioactive compounds [21,22], and these include the so-called standard markers such as phenolic caffeic acid derivatives, alkylamides, and polysaccharides, all of which have been proposed to be responsible for the purported medical benefits of various Echinacea species extracts [29]. However our recent studies on different types of Echinacea extract suggest that specific bioactivities may not be attributed to a single component. In addition EF, like other Echinacea-derived extracts, contains numerous other bioactive compounds such as flavonoids and alkaloids [29], and it is conceivable that the key to the relatively high potency of EF is the particular combination or balance of individual ingredients. Recent studies on the Mediterranean herb Cistus incanus (rock rose) provide some interesting comparisons. Thus a polyphenol-rich Cistus extract showed similar anti-IV activities to those described in this report, suggesting a similar mode of action [18]. The mechanism of Cistus anti-viral activity was not elucidated however, so a comparative study of these two extracts could be useful and provide interesting implications for the design of effective anti-IV compounds.
In contrast, the study of Palamara et al [16] showed that an individual polyphenol, resveratrol, a common constit-uent of red grapes and various other plants, could inhibit IV replication by interfering with signaling pathways involved in viral RNP translocation. Thus an appropriate combination of plant polyphenols could provide a multifunctional approach to the control of influenza virus replication and its associated symptoms.

Conclusion
The data presented in this work have shown that a standardized preparation of Echinacae has the potential to impair influenza virus propagation, including seasonal strains and strains of highly pathogenic avian influenza viruses as well as the new pandemic strain of swine origin at concentrations recommended for oral use and below. Furthermore the preparation does not induce emergence of resistant virus variants and is still active against strains that have become resistant to treatment with neuraminidase inhibitors. This potential, the availability and the lack of toxicity make this preparation an interesting option in the control and treatment of influenza virus infections

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-(4,5dimethylthiazol-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% CO 2 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).

MIC 100 values
MIC 100 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% CO 2 until viral CPE were complete in control wells containing untreated virus (usually 2 days). Additional wells contained cells not exposed to virus. The MIC 100 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.

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% CO 2 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 preincubations 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. 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