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)

Abstract

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.

Figure 1

MIC 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 prevention of CPE.

Figure 2

EF 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 performed in triplicate, and titrations in duplicate.

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.

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: (-) 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.

Figure 5

EF treatment does not select for resistant IV variants. 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.