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Anti-viral properties and mode of action of standardized Echinacea purpurea extract against highly pathogenic… Pleschka, Stephan; Stein, Michael; Schoop, Roland; Hudson, James B Nov 13, 2009

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ralssBioMed CentVirology JournalOpen AcceResearchAnti-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)Stephan Pleschka*1, Michael Stein1, Roland Schoop2 and James B Hudson3Address: 1Institute for Medical Virology, Justus-Liebig-University Giessen, Frankfurterstr. 107, D-35392 Giessen, Germany, 2Bioforce AG, Gruenaustr, CH-9325 Roggwil, Switzerland and 3Department of Pathology & Laboratory Medicine, University of British Columbia, 2733 Heather Street, Vancouver V5Z 3J5, CanadaEmail: Stephan Pleschka* - stephan.pleschka@mikro.bio.uni-giessen.de; Michael Stein - Michael.Stein@viro.med.uni-giessen.de; Roland Schoop - r.schoop@bioforce.ch; James B Hudson - jbhudson@interchange.ubc.ca* Corresponding author    AbstractBackground: Influenza virus (IV) infections are a major threat to human welfare and animal healthworldwide. Anti-viral therapy includes vaccines and a few anti-viral drugs. However vaccines arenot always available in time, as demonstrated by the emergence of the new 2009 H1N1-typepandemic strain of swine origin (S-OIV) in April 2009, and the acquisition of resistance toneuraminidase inhibitors such as Tamiflu® (oseltamivir) is a potential problem. Therefore theprospects for the control of IV by existing anti-viral drugs are limited. As an alternative approachto the common anti-virals we studied in more detail a commercial standardized extract of thewidely 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, aswell as swine origin IV (S-OIV, H1N1), were all inactivated in cell culture assays by the EFpreparation at concentrations ranging from the recommended dose for oral consumption toseveral orders of magnitude lower. Detailed studies with the H5N1 HPAIV strain indicated thatdirect contact between EF and virus was required, prior to infection, in order to obtain maximuminhibition in virus replication. Hemagglutination assays showed that the extract inhibited thereceptor binding activity of the virus, suggesting that the extract interferes with the viral entry intocells. 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 uponpassaging. Furthermore, the Tamiflu®-resistant virus was just as susceptible to EF as the wild typevirus.Conclusion: As a result of these investigations, we believe that this standard Echinaceapreparation, used at the recommended dose for oral consumption, could be a useful, readilyavailable and affordable addition to existing control options for IV replication and dissemination.Published: 13 November 2009Virology Journal 2009, 6:197 doi:10.1186/1743-422X-6-197Received: 9 September 2009Accepted: 13 November 2009This article is available from: http://www.virologyj.com/content/6/1/197© 2009 Pleschka et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 9(page number not for citation purposes)Virology Journal 2009, 6:197 http://www.virologyj.com/content/6/1/197BackgroundInfluenza viruses (IV) continue to cause problems glo-bally in humans and their livestock, particularly poultryand pigs, as a consequence of antigenic drift and shift,resulting frequently and unpredictably in novel mutantand re-assortant strains, some of which acquire the abilityto cross species barriers and become pathogenic in theirnew hosts [1]. Prospects for the emergence of pandemicstrains of swine and avian origin have been discussed inseveral recent reports [2,3]. Some of the highly pathogenicavian IV (HPAIV) strains, in particular H5N1, have occa-sionally infected humans and pose a severe threat becauseof their high pathogenicity, with mortality rates exceeding60% [4,5].The practicality and efficacy of control by timely vaccina-tion has been questioned [1,6,7], and potential control ofIV by synthetic anti-viral chemicals has usually beenthwarted by the inevitable emergence of resistant strains,a situation that has been documented in the case of theM2 ion-channel inhibitors, such as adamantane deriva-tives, and the neuraminidase inhibitors such as oseltami-vir and zanamivir [8,9]. Virus-strain specificity is anotherlimitation in the use of these inhibitors.Alternative approaches to therapy that overcome theseobstacles are urgently needed and have been suggested.These include manipulation of specific signaling path-ways known to be involved in virus replication [10,11]. Assuch, the Raf/MEK/ERK-signal transduction cascade andactivation of the transcription factor NF-κB were shown tobe essential for efficient nuclear export of the viral ribonu-cleoprotein (RNP) complexes. They have proven to behighly interesting targets, as their inhibition significantlyreduces virus replication without emergence of resistantvariants in vitro and in vivo [12-15]. Another approach isthe use of broad-spectrum and chemically-standardizedanti-IV herbal extracts and compounds with demon-strated efficacy in vitro [16-19]. These could conceivablyafford a more generalized inhibition of all virus strains,either by virtue of inactivating the virus directly or byinterfering with one or more essential stages in virus rep-lication or dissemination. Furthermore anti-viral herbalextracts frequently exhibit multiple bioactivities [20], andthis could enable their use at relatively low doses of theactive compounds, possibly acting in synergy, while at thesame time providing a relatively safe "drug" with few sideeffects. Needless to say, acquisition of resistance to herbalcompounds is also a potential problem; consequently thiswould need to be evaluated, although if multiple bioac-tive compounds were involved, this would substantiallyreduce the risk of resistant viruses emerging.EF), which has become a very popular herbal "remedy"for the symptoms of "colds and flu". In addition to pos-sessing potent virucidal activity against several membranecontaining viruses, including H3N2-type IV, at the recom-mended dose for oral consumption, the preparation alsoeffectively reversed virus-induced pro-inflammatoryresponses in cultured epithelial cells [21]. Some Echina-cea-derived preparations also possess selective anti-bacte-rial and immune modulation activities that might alsocontribute to their beneficial properties [22,23]. However,our studies also indicated that anti-viral and cytokine-inhibitory properties vary widely among different Echina-cea species and components [24-26]; thus it is importantto carry out research on Echinacea preparations that havebeen standardized and chemically characterized.The objective of the present study was to investigate theanti-IV activity in more detail, and to elucidate possiblemechanisms of action on a variety of IV strains (humanand avian), with emphasis on a human isolate of theH5N1-type HPAIV, and to evaluate the potential for emer-gence of resistant strains, in comparison with oseltamivir(Tamiflu®).ResultsEchinaforce® (EF) and Virus ConcentrationWe reported previously that at concentrations up to 1.6mg/ml (dry mass/vol, the recommended oral dose) the EFextract showed no apparent cytotoxic effects, according totrypan blue staining, MTT assays, or microscopic examina-tion [[21], data not shown]. However at concentrations of>1.6 μg/ml ≥99% inactivation of H3N2-type IV wasachieved (Table 1). The degree of inactivation dependedon the virus dose, as might be expected (Fig. 1). MIC100values increased from 0.32 μg/ml for 102 PFU/ml virus, upto 7.5 μg/ml for 105 PFU/ml.In order to exclude the possibility that the virucidal effectmight be subtype specific or related only to human IV, weanalyzed the effect of EF in non toxic concentrations on ahuman isolate of a H5N1-type HPAIV (KAN-1). Virusyield reduction assays were carried out with KAN-1, whichhad been pre-incubated with various concentrations ofEF, from 0.1 to 50 μg/ml (Fig. 2). At the highest concen-tration the yield was reduced by more than 3 log10. Fur-thermore we tested the inhibitory effect of EF on humanH1N1-type (PR8) and a H7-type HPAIV (FPV) andobtained comparable results (data not shown), indicatingthat EF affects not only human IV (H3N2, H1N1) but alsoboth types (H5, H7) of HPAIV (data not shown).Time of addition of Echinaforce®All the IV strains tested, the human pathogenic VictoriaPage 2 of 9(page number not for citation purposes)We recently reported the anti-viral properties of a stand-ardized preparation of Echinacea purpurea (Echinaforce®,(H3N2), PR8 (H1N1), S-OIV (H1N1), and the avianstrains KAN-1 (H5N1) and FPV (H7N7) were susceptibleVirology Journal 2009, 6:197 http://www.virologyj.com/content/6/1/197to the EF, but only as a result of direct contact. Pre-incuba-tion of cells with extract, followed by virus infection, orpost-exposure of the infected cells to EF, inhibited virusreplication to a lesser extent (data not shown). To investi-gate this in more detail, several experiments were per-formed with KAN-1 to determine the effect of adding EFat different times relative to virus infection of the cells.Complete inhibition was achieved by incubating KAN-1and 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 partialreduction in virus production, compared to untreated(lane 1). These results suggest that EF was acting eitherdirectly on the virus or at a very early stage in the replica-tion cycle. It is noteworthy to mention, that removal of EFcontaining medium 6.5 hours p.i. and further incubationin normal medium for 1.5 hours in order to prevent anexposure of newly formed virions to EF prior to titration,did not change this result.Intra-cellular RNP localizationNext, the production and intra-cellular localization ofviral RNP were determined by immunofluorescence, inMDCK cells infected with KAN-1, with and without EFtreatment (Fig 4). In normally infected cells (- EF), thenucleocapsid protein (NP, green), which is the main com-ponent of the RNPs, appeared initially in the nucleus (6hours) followed by migration to the cytoplasm (8 hours).The same pattern was seen in EF-treated cells infected withuntreated virus (cells + EF), and in cells exposed to EF afterinfection (EF p.i.). However, when cells were infected withEF-treated virus (virus + EF), the overall number of posi-tive cells was significantly reduced. Nevertheless, theamount and the localization of RNPs detected in cellsinfected with pre-treated IV was the same as for untreatedcells infected with untreated virus. It should be noted thatthe 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 avery early stage before replication, but once the virus hasentered the cells its replication and spread are not affected.MIC depends on the viral doseFigur  1MIC depends on the viral dose. Increasing amounts of IV (Victoria, H3N2) were used to determine the MIC100 of EF. Serial dilutions of EF, in quadruplicate, were incubated with the amounts of IV indicated (102, 103, 104, 105 PFU), and transferred to cells for CPE-endpoint determination, as described in Materials and Methods section. The MIC100 (μg/ml) is the concentration of EF that leads to complete preven-tion of CPE.Table 1: Anti-influenza virus (H3N2) effect of EFEF dilution(μg/ml)Virus titer(PFU, % of control)1:30 (53.3) < 0.11:102 (16) < 0.11: 103 (1.6) < 0.11: 104 (0.16) 1.0 ± 01: 105 (0.016) 110 ± 7.8EF acts in a dose dependent mannerFigure 2EF acts in a dose dependent manner. H5N1 HPAIV (MOI = 0.001) and MDCK cells were pre-incubated with EF at the indicated concentrations 1 hour prior to infection. Infected cells were then incubated in media with EF at the appropriate concentrations for 24 hours and the infectious titer was determined (FFU/ml). The experiment was per-formed in triplicate, and titrations in duplicate.0123456789Control 0,1 μg/ml 1,0 μg/ml 10 μg/ml 50 μg/mlEF concentrationvirus titer FFU/ml (106)Page 3 of 9(page number not for citation purposes)Interaction of Echinaforce® with Viral HAThe first step in entry of IV into cells depends on the inter-action between the viral HA and a specific cellular sialicAliquots of H3N2 virus, containing 105 PFU/ml, were incubated at 22°C for 60 min with the indicated concentrations of EF, and assayed for remaining PFU/mlVirology Journal 2009, 6:197 http://www.virologyj.com/content/6/1/197acid containing receptor. If EF could inhibit this interac-tion by binding to the HA, then entry of virus might beprevented. Receptor binding of functional HA can bemeasured by its ability to agglutinate chicken erythro-cytes, which can be easily enumerated visually. Directinteraction between virus and EF was therefore examinedby inspecting viral hemagglutination (HA) activity in thepresence and absence of EF. Results for the pandemic S-OIV (H1N1) and two HPAIV (H5, H7) are shown in Table2. EF inhibited HA activity for all 3 virus strains, in a con-centration and time-dependent manner. The same con-centrations of EF without virus showed nohemagglutination, as expected (data not shown). In addi-ity was due to an interference by EF. As this is effectiveagainst different human and avian strains, EF might exertan unspecific effect on IV replication by interfering withviral receptor binding and entry.Lack of Resistance to Echinaforce®Treatment with currently available anti-influenza drugsdirectly targeting the virus has the drawback that, due tothe high mutation rate of IV, resistant strains will inevita-bly arise. This has been shown for neuraminidase inhibi-tors like Tamiflu® in regard to seasonal IV, H5N1 HPAIVand in recent reports for the pandemic S-OIV[2,3,9,27,28]. Therefore, any competitive alternativePre-treatment of IV with EF is most effectiveFigu  3Pre-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.--++++-Incubation of cells p.i.+-+-+--Pre-incubation cells-+-++--Pre-incubationvirus7654321Treatment01020304050607080901001 2 3 4 5 6 7 1 2 3 4 5 6 78h p.i. 24h p.i.virustiter(%)Page 4 of 9(page number not for citation purposes)tion there was no visual evidence of erythrocyte lysis inany of the reactions. Therefore the inhibition in HA activ-should have the advantage of preventing emergence ofresistant IV variants [11]. This might be different for a sub-Virology Journal 2009, 6:197 http://www.virologyj.com/content/6/1/197stance that unspecifically blocks virus activity. The possi-bility of emergence of EF-resistant virus was evaluated bycomparing relative H5N1 virus yields in the presence andabsence of EF, or Tamiflu®, during consecutive passagesthrough cell cultures. Results are shown in Fig 5. After oneround of replication virus yields were substantiallyreduced by 50 μg/ml EF or 2 μM Tamiflu®. However inrounds 2 and 3 the yields in the presence of Tamiflu® weresimilar to controls, indicative of emergence of resistantvirus variants, whereas in the presence of EF yields contin-ually remained low, indicating lack of EF-resistant virus.To determine if Tamiflu®-resistant virus remained sensi-tive to EF, the growth of Tamiflu®-resistant virus (pro-duced in the above experiments) was tested in thepresence and absence of EF. EF (50 μg/ml) reduced theyield of Tamiflu®-resistant virus by more than 3 log10 viralFFU, similar to that of standard virus (data not shown).DiscussionThese results have shown that Echinaforce® (EF), a stand-ardized Echinacea purpurea extract, has potent anti-viralactivity against all the IV strains tested, namely humanVictoria (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 rec-ommended dose for oral consumption, to as little as 1.6μg/ml of the extract, a 1:1000 dilution, could inactivatemore than 99% of virus infectivity, and treated virus gaverise to markedly reduced yields of virus in cell culture.However, direct contact between virus and EF wasrequired for this inhibitory effect, since pre-treatment ofcells before virus infection, or exposure of cells p.i. to EF,led to substantially less inhibition, indicating that theanti-viral effect was manifest at a very early stage in theinfection process. This was then confirmed by the use ofhemagglutination assays, which clearly showed that EFinhibited HA activity and consequently would block entryof treated virus into the cells. Nevertheless, the mecha-nism of this inhibition needs to be studied in more detail.The general inhibition of EF against the different virusstrains constitutes a significant advantage over other strainspecific anti-virals, such as adamantanes [8,9]. Further-more, the lack of emergence of EF-resistant viruses duringsequential passage is a significant advantage over Tami-flu®, which under similar culture conditions readilyallowed resistant virus strains to develop. In addition theTamiflu®-resistant virus was still very sensitive to EF. Theseresults indicate that EF could be helpful in IV control, andwould be complemented by the known ability of EF tocounteract pro-inflammatory cytokine and chemokineinduction caused by IV and other viruses, as well as theselective anti-bacterial activities of Echinacea extracts [23].Thus EF could play a multi-functional role during IV infec-tions.Intra-cellular RNP production and localization is not affected by EFFigure 4Intra-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 fol-lows: (-) EF, normal infection with no EF treatment; (EF p.i.), infected cells treated with EF (50 μg/ml) after infection; virus (+) EF, virus pretreated with EF (50 μg/ml); cells (+) EF, cells pretreated with EF (50 μg/ml), and infected with untreated virus. Infected cells were then incubated in medium with or without EF for 6 and 8 hours and the intra-cellular amount and localization of viral RNPs (green), as well as the nuclei (blue), were detected by immunofluorescence.6h p.i.8h p.i.(-) EF EF p.i. Virus (+) EF Cells (+) EFTable 2: Interaction of EF with viral HAμg/ml EF 1 hour μg/ml EF 4 hoursIV strainPos. Ctrl.Neg. Ctrl.50 100 200 400 800 50 100 200 400 800S-OIV (H1N1) + - - - - - - - - - - -KAN-1 (H5N1) + - + + + - - - - - - -FPV (H7N7) + - + + + - - - - - - -Page 5 of 9(page number not for citation purposes)+: indicates hemagglutinin activity (agglutination of erythrocytes)-: indicates no hemagglutin activity. Ctrl, controlVirology Journal 2009, 6:197 http://www.virologyj.com/content/6/1/197The Echinaforce® extract contains known concentrationsof potentially bioactive compounds [21,22], and theseinclude the so-called standard markers such as phenoliccaffeic acid derivatives, alkylamides, and polysaccharides,all of which have been proposed to be responsible for thepurported medical benefits of various Echinacea speciesextracts [29]. However our recent studies on differenttypes of Echinacea extract suggest that specific bioactivitiesmay not be attributed to a single component. In additionEF, like other Echinacea-derived extracts, contains numer-ous other bioactive compounds such as flavonoids andalkaloids [29], and it is conceivable that the key to the rel-atively high potency of EF is the particular combination orbalance of individual ingredients.Recent studies on the Mediterranean herb Cistus incanus(rock rose) provide some interesting comparisons. Thus apolyphenol-rich Cistus extract showed similar anti-IVactivities to those described in this report, suggesting asimilar mode of action [18]. The mechanism of Cistusanti-viral activity was not elucidated however, so a com-parative study of these two extracts could be useful andprovide interesting implications for the design of effectiveanti-IV compounds.uent of red grapes and various other plants, could inhibitIV replication by interfering with signaling pathwaysinvolved in viral RNP translocation. Thus an appropriatecombination of plant polyphenols could provide a multi-functional approach to the control of influenza virus rep-lication and its associated symptoms.ConclusionThe data presented in this work have shown that a stand-ardized preparation of Echinacae has the potential toimpair influenza virus propagation, including seasonalstrains and strains of highly pathogenic avian influenzaviruses as well as the new pandemic strain of swine originat concentrations recommended for oral use and below.Furthermore the preparation does not induce emergenceof resistant virus variants and is still active against strainsthat have become resistant to treatment with neuramini-dase inhibitors. This potential, the availability and thelack of toxicity make this preparation an interestingoption in the control and treatment of influenza virusinfectionsMethodsStandard Echinacea PreparationEchinaforce® (obtained from A. Vogel Bioforce AG, Rog-gwil, Switzerland) is a standardized preparation derivedby ethanol extraction of freshly harvested Echinacea purpu-rea herb and roots (95:5). The composition of markercompounds (ie. those compounds known to characterizethis species of Echinacea) was described previously [21].The concentration of ethanol was 65% v/v. The final con-centration of ethanol in the experimental reactions andcultures was too low to cause adverse effects on the cellsor viruses. In addition the preparation was free of detecta-ble endotoxin (as determined by means of a commercialassay kit, Lonza Walkersville Inc., MD, lower limit ofdetection 0.1 unit/ml), and the administered amount thatwas effective in our experiments, up to the recommendedoral dose of 1.6 mg/ml, was not cytotoxic according totrypan blue staining, MTT formazan assays (MTT = 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan), andmicroscopic examination [[21], and data not shown].Cell lines & VirusesMadin-Darby canine kidney cells (MDCK) were acquiredoriginally from ATCC and were passaged in Dulbecco'sMEM (DMEM), in cell culture flasks, supplemented with5-10% fetal bovine serum, at 37°C in a 5% CO2 atmos-phere (cell culture reagents were obtained from Invitro-gen, Ontario (CA) or Karlsruhe (DE)). No antibiotics oranti-mycotic agents were used for experiments performedin the Hudson laboratory. In the Pleschka laboratory thecell cultures were also grown in DMEM, 10% FCS but sup-EF treatment does not select for resistant IV variantsFigure 5EF treatment does not select for resistant IV vari-ants. MDCK cells were infected with KAN-1 (MOI = 0.001) and incubated 24 hours either with media without EF (black bars), or containing EF (50 μg/ml hatched bars) or Tamiflu® (2 μM, grey bars). Supernatant was titrated by FFU assay and used for a second round of infection of fresh MDCK cells. Three passages (1st, 2nd, 3rd round) were performed and after each the virus titer (FFU/ml) was determined by FFU assay. FFU titres of EF- and Tamiflu®-treated samples were calculated as percentage of controls set at 100%. Shown is the mean of duplicate experiments titrated in duplicates.0204060801001201. Round 2. Round 3. RoundVirus titer(%)Control EF TamifluPage 6 of 9(page number not for citation purposes)In contrast, the study of Palamara et al [16] showed thatan individual polyphenol, resveratrol, a common constit-plemented by 100 U/ml penicillin and 100 μg/ml strepto-mycin (P/S).Virology Journal 2009, 6:197 http://www.virologyj.com/content/6/1/197The following influenza A virus strains were used: humanstrain A/Victoria/3/75 (Victoria, H3N2) acquired from theBC Centre for Disease Control, Vancouver. The humanHPAIV 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 thehuman strain A/Puerto Rico/8/34 (PR8, H1N1) wereobtained from the IV strain collection in Giessen, Ger-many; the human isolate of the 2009 pandemic IV ofswine-origin A/Hamburg/1/09 (S-OIV, H1N1) was pro-vided to S. Pleschka by M. Matrosovich, Marburg, Ger-many. KAN-1 and FPV or PR8 were propagated on MDCKcells with low serum but without trypsin (KAN-1, FPV) orin embryonated chicken eggs (PR8), respectively. All otherstrains were propagated on MDCK cells in the presence oftrypsin (2.5 μg/ml). Stock viruses were prepared as clari-fied cell-free supernatants or allantois fluid, respectively,with titers ranging from 107 to 108 PFU (plaque-formingunits) per ml. and stored at -75°C. Strains were titratedeither by standard plaque assay or by focus forming assays(see below).MIC100 valuesMIC100 values of EF were determined from CPE-endpointassays, as follows: The Echinacea extract, in 200 μl aliq-uots, was serially diluted two-fold across replicate rows ofa 96-well tray, in medium, starting at the recommendedoral dose of 1.6 mg/ml. Virus, 100 PFU in 100 μl, wasadded to each well and allowed to interact with the extractfor 60 min at 22°C. Following the incubation period, themixtures were transferred to another tray of cells fromwhich the medium had been aspirated. These trays werethen incubated at 37°C, 5% CO2 until viral CPE werecomplete in control wells containing untreated virus (usu-ally 2 days). Additional wells contained cells not exposedto virus. The MIC100 was the maximum dilution at whichCPE was completely inhibited by the extract. In mostassays the replicate rows gave identical end-points; whentwo-fold differences were encountered arithmetic meansand standard deviations were calculated. In the alternative(intra-cellular) method, the cells were incubated with thediluted extracts first, before adding virus.Virus titrationsStrain Victoria (H3N2) was titrated by standard plaqueassay techniques in MDCK cells with agarose overlays. Theother strains were assayed by focus formation in MDCKcells as follows: Cells were grown overnight (to 90% con-fluency) in complete medium in 96-well trays, washedand inoculated with 50 μl of serially diluted (10-1 to 10-8)virus in PBS containing 0.2% BA, 1 mM MgCl2, 0.9 mMCaCl2, 100 U/ml penicillin and 0.1 mg/ml streptomycin(PBS/BA), for 60 min at room temperature. The inoculumCO2 for 44 hours. To detect foci of infection the cells werepermeabilized with 330 μl fixing solution (4% parafor-maldehyde, 1% triton X-100, in PBS) and stored at 4°Cfor 60 min followed by 3 washes with PBS/0.05% Tween20, 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 incu-bated with 2nd antibody (anti-mouse HRP antibody SantaCruz sc2005) diluted in PBS/3% BA at room temperaturefor 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) for60 min followed by washing in dH2O. Foci were scannedand analyzed by means of Photoshop software (Adobe).All titrations were performed in duplicate.Pre-incubationsIn some experiments aliquots of virus (H3N2 or H5N1) inPBS/BA or the cells in complete medium, were pre-incu-bated with EF (50 μg/ml) at room temperature or 37°Crespectively for 60 min, prior to infection. Infected cellsand controls were then incubated in medium containingEF (50 μg/ml) at 37°C, 5% CO2 for 24 hours, at whichtime supernatants were removed for focus assays.Intra-cellular RNP localizationCells were grown and infected on cover slips, and pre-incubations of the viruses were carried out as describedabove. 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 wascarried out at room temperature for 60 min in the dark.Cells were washed again and incubated with DAPI (0.1mg/ml PBS/3%BA, Roth Germany) for 10 min in the darkto stain nuclei. After further washing the cover slips withcells 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. Cellswere examined and digitized with a TCS SP5 confocallaser scanning microscope (Leica, Germany).Hemagglutination assay25 μl EF in PBS at the indicated concentrations wereadded to wells of a 96-well tray. Thereafter 25 μl of viruswith ca. 2560 HAU/ml were added. The plates were incu-bated for 60 min at 4°C. After this incubation period, 50μl of chicken erythrocyte suspension (CES, 0.5% in PBS)were added to each well. The plates were further incubatedfor 60 min or 4 hours at 4°C. Wells were visuallyinspected for the presence or absence of hemagglutina-tion. Positive and negative controls without EF treatmentPage 7 of 9(page number not for citation purposes)was replaced by 150 μl MC media (1× DMEM, BA, P/S,1.5% methyl cellulose). Cells were incubated at 37°C, 5%or without virus were included. To assay possible hemag-glutination by EF itself, 50 μl of EF in PBS at the indicatedVirology Journal 2009, 6:197 http://www.virologyj.com/content/6/1/197concentrations were incubated with 50 μl CES for 60 minor 4 hours at 4°C. All assays were performed in quadrupli-cate.Virus Resistance AssayMDCK cells grown over night at 37°C and 5% CO2 werepre-incubated with 2 ml complete medium (1× DMEM,10% FCS, Pen/Strep) with or without EF (50 μg/ml), at37°C and 5% CO2 for 60 min. In parallel virus in PBS/BAwas incubated with EF (50 μg/ml) or left untreated for 60min. After the pre-incubation period the cells werewashed and infected with 500 μl virus suspension (MOI =0,001) (+/-) Echinaforce® (50 μg/ml). Cells were thenincubated for 60 min in the dark at room temperatureafter which the inoculum was removed. Cells were furtherincubated in 2 ml medium (DMEM/BA/P/S with Echina-force® (50 μg/ml), Tamiflu® (2 μM) or without test sub-stances) at 37°C, 5% CO2 for 24 hours. Samples of thesupernatants were collected, which were then assayed byfocus forming assay for further determination of infec-tious virus. Following the assays, these supernatants wereused to infect another set of cultures under the same con-ditions as described above. This process of sequentialinfection with supernatants was repeated once more toyield in total three rounds of infection and replication.Experiments done in duplicates were stopped when theTamiflu® sample reached titers of the untreated control.BiosafetyAll experiments with infectious virus were performedaccording to German and Canadian regulations for thepropagation of influenza A viruses. All experimentsinvolving highly pathogenic influenza A viruses and thepandemic S-OIV were performed in a biosafety level 3(BSL3) containment laboratory approved for such use bythe local authorities (RP, Giessen, Germany).AbbreviationsCPE: cytopathic effects; EF: Echinaforce®; FFU: focus-form-ing unit; HA: hemagglutinin; HAU: hemagglutinatingunits; IV: influenza virus; PFU: plaque-forming unit; RNP:(viral) ribo-nucleoprotein; S-OIV: swine-origin influenzavirus.Competing interestsThe work was in part financially supported by Bioforce AG(to S.P and J.H.). There were no competing interests.Authors' contributionsSP directed and participated in the studies on avian andH1N1 viruses, and co-wrote the manuscript.MS carried out the experimental work in Germany.RS organized the overall project, supplied the standard-ized source material, and helped edit the manuscript.JH carried out the experimental work in Canada, and co-wrote and edited the manuscript.AcknowledgementsWe would like to thank E. Lenz for excellent technical assistance. This work was supported in part by grants of the European Specific Targeted Research Project „EuroFlu - Molecular Factors and Mechanisms of Transmission and Path-ogenicity of Highly Pathogenic Avian Influenza Virus” funded by the 6th Frame-work Program (FP6) of the EU (SP5B-CT-2007-044098, to S.P) and the "FluResearchNet - Molecular Signatures determining Pathogenicity and Species Transmission of Influenza A Viruses" (01 KI 07136, to S.P.)References1. 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Echinacea pallida (Nutt.)Nutt., Echinacea purpurea (L.) Moench: a review of theirchemistry, pharmacology and clinical properties.  J PharmPharmacol 2005, 57:929-954.yours — you keep the copyrightSubmit your manuscript here:http://www.biomedcentral.com/info/publishing_adv.aspBioMedcentralPage 9 of 9(page number not for citation purposes)


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