In vitro antiviral activity of Echinaforce ® , an Echinacea purpurea preparation, against common cold coronavirus 229E and highly pathogenic MERS-CoV and SARS-CoV

Background: Coronaviruses (CoVs) were long thought to only cause mild respiratory and gastrointestinal symptoms in humans but recent outbreaks of Middle Eastern Respiratory Syndrome (MERS)-CoV, Severe Acute Respiratory Syndrome (SARS)-CoV, and the newly identified SARS-CoV-2 have cemented their zoonotic potential and their capacity to cause serious morbidity and mortality, with case fatality rates ranging from 2 to 35%. Currently, no specific prophylaxis or treatment is available for CoV infections and therefore we investigated the antiviral potential of Echinacea purpurea (Echinaforce ® ) against human coronavirus (HCoV) 229E and the highly pathogenic MERS- and SARS-CoVs in vitro. Methods: To evaluate the antiviral potential of Echinaforce we pre-treated virus particles and cells and evaluated remaining infectivity by limited dilution. Furthermore, we exposed cells to the extract post-infection to further estimate its potential as a prophylaxis and treatment against coronaviruses. We also determined the protective effect of Echinaforce in reconstituted nasal epithelium.


Conclusions:
These results suggest that Echinacea purpurea preparations, such as Echinaforce, could be effective as prophylactic treatment for all CoVs, including newly occurring strains, such as SARS-CoV-2.

Background
Coronaviruses (CoVs) are believed to be responsible for 10-15% of all upper respiratory tract infections in humans and were mainly thought to be responsible for the common cold until 2002 (1). Currently, seven CoVs have been found to cause disease in humans. Four of those, HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1, are non-zoonotic and cause worldwide outbreaks predominantly in the winter period. These HCoVs replicate in the nasopharynx and generally cause mild, self-limited upper respiratory tract infections with short incubation periods, although lower tract respiratory infections and pneumonia have occasionally been described (2)(3)(4)(5). The more virulent coronaviruses, Middle East respiratory syndrome (MERS)-CoV and Severe Acute Respiratory Syndrome (SARS)-CoV have animal reservoirs with proposed origins in bats (6) and can cause severe pneumonias with longer incubation periods and often fatal outcome (7). SARS-CoV was introduced into the human species in 2002 causing a worldwide epidemic in 2003 culminating in 8422 infections and 916 deaths (8). MERS-CoV is heavily endemic in dromedary camels and leads to lower respiratory tract infections in humans with a current case-fatality rate of 35.5% (9). As of December 31 st 2019, a pneumonia outbreak originating from a live seafood market in Wuhan, China, has resulted in an increasing number of fatal severe respiratory tract infections and, so far unprecedented, travel bans (10). To date, there is a lack of established and clinically tested antiviral compounds against coronaviruses in general and, more distressingly, the zoonotic betacoronaviruses (11). Given their increasing incidence and burden, an inexpensive, accessible and effective treatment for HCoVs is of utmost importance.
Echinacea plants have traditionally been used in North America for the prevention and treatment of cold and flu symptoms and are now one of the most widely used medical plants in both North America and Europe (12). Several different products are on the market, not only varying in the Echinacea species and the parts of the plant used but also in manufacturing procedures, which, unfortunately, results in a large variability in quality and activity (13,14). Echinaforce ® is a standardized preparation extracted from herb and roots of freshly harvested Echinacae purpurea plants with a 65% alcoholic solution.
Echinaforce as prevention and treatment of respiratory tract infections has been investigated in both pre-clinical and clinical studies and its beneficial effects documented (15)(16)(17)(18). Specific mechanism of action is not fully understood but in vitro studies indicate that Echinaforce inhibits membranous respiratory viruses including influenza A and B, respiratory syncytial virus (RSV) or parainfluenza virus, through direct interaction with virus particles and viral envelope proteins (19,20). Intracellular activity of Echinacea has been observed for some viruses (e.g. influenza and herpes simplex virus) but not others (e.g. RSV), and only at higher concentrations required for extracellular inactivation. Furthermore, Echinacea has been shown to interfere with virus mediated cytokine release (21,22) and since typical symptoms of the common cold, sneezing, coughing and runny nose, are the results of the stimulation of pro-inflammatory cytokines, the reduction of cytokine release might help to ease symptoms. In a randomized, double-blind, multicenter, non-inferiority clinical trial Echinaforce was demonstrated to be non-inferior to Oseltamivir in patients with influenza-like illness and confirmed influenza infection with a trend for lower incidence of complications with Echinaforce Hot Drink as with Oseltamivir (16).
The antiviral activity of Echinacea has been investigated in vitro for most of the respiratory viruses associated with common colds and flu, but as of yet, not for coronaviruses. HCoV-229E is a typical representative of a coronavirus strain causing a seasonal common cold. By using HCoV-229E as a model, we investigated the anticoronaviral activity of Echinaforce extract, thereby closing the knowledge gap on the antiviral effects of Echinacea purpurea on typical common cold viruses. Furthermore, we expanded our analysis to the highly pathogenic SARS-and MERS-CoVs and other viruses that cause disease in humans. Additionally, we utilized an organotypic respiratory cell culture system (MucilAir™) of nasal origin to investigate the protective effect of Echinaforce against coronaviruses in a culture system that closely mimics in vivo human airway epithelium. In the current study, we observed an irreversible inhibition of the infectivity of three coronavirus strains upon direct contact with Echinaforce. Furthermore, a protective effect was observed upon pre-treatment in an organotypic airway model.

Echinacea preparation
Echinaforce® (A.Vogel AG, Roggwil, Switzerland) is derived from hydroethanolic extraction (65% v/v ethanol) of freshly harvested Echinacea purpurea herb and roots (95:5). The composition of typical marker compounds such as caftaric acid, chlorogenic acid, echinacoside and alkylamide derivates has been previously described elsewhere (20). The final concentration of ethanol in the extract was 65% v/v with 16 mg/ml dry mass Echinacea. Experimental concentrations are expressed as dry mass of Echinaforce extract.  Germany) at 800 g for 15 min. Viruses were recovered from the Ultrafiltration Unit with glycine buffer (3750 mg/l glycine, 10 g/l beef extract, 14.6 g/l NaCl, pH 9.5, Sigma-Aldrich, Germany), and diluted in 1:10 in 5%-FBS DMEM. Residual virus infectivity was determined by a limiting dilution assay (TCID 50 ) according to Spearman-Karber (25).

Pre-treatment of cells
Huh-7 cells were incubated with 0, 1, 10 or 50 µg/ml Echinaforce in cell culture medium for 3 days at 33 °C. Thereafter, Echinaforce-containing medium was removed and cells infected with 100 TCID 50 HCoV-229E for 1 h at 33 °C. Medium was replaced and cells further incubated for 48 h at 33 °C and virus titer determined by limiting dilution assay.  TCID 50 /ml HCoV-229E was exposed to increasing concentrations of extract and the effect on virus infectivity determined by quantifying infectious virus particles by a limiting dilution assay. Exposure to Echinaforce for 60 minutes led to a dose dependent reduction of infectious HCoV-229E virus particles (Fig. 1). Complete inhibition of replicating virus was observed at 50-100 µg/ml extract, with mean inhibitory concentration (IC 50 ) 3.2 ug/ml. Parallel incubation of cells with Echinaforce showed stable cell viability at all tested concentrations (Fig. 1). Results show, that upon contact with the extract, a permanent alteration of virus infectivity occurred, as the inhibitory effect could not be reversed through extensive washing of treated virus particles (Fig. 2a). In contrast, pre-treatment of cells had no influence on HCoV-229E infectivity or replication (Fig. 2b). In cells treated post-infection, a small reduction in virus titer was observed after treatment with the highest dose of 50 µg/ml (Fig. 2c).

Echinaforce inhibits HCoV-229E infection of respiratory epithelial cells
To evaluate how Echinaforce may exert its antiviral activity in a more natural setting, we utilized a re-differentiated, pseudostratified respiratory epithelial cell culture model. The reconstituted epithelium is functional, produces mucus and exhibits active ciliary-beating and mucociliary clearance much like in vivo epithelium. To simulate daily usage of the extract, cultures were pre-treated with 0, 10 and 50 µg/ml Echinaforce for one day. Virus exposure, reflecting common cold transmission, was simulated by dropwise application of 100 TCID 50 HCoV-229E virus suspension onto the apical surface of the epithelium, covered with 0, 10 and 50 µg/ml Echinaforce (Fig. 3a). Virus infection and replication was analyzed

Echinaforce exerts antiviral effects on MERS and SARS coronaviruses
Since Echinacea preparations have shown an antiviral effect against HCoV-229E and other membranous respiratory viruses (12,26), we expected to see a similar effect on the related, highly pathogenic coronaviruses MERS-CoV and SARS-CoV. To this end, we evaluated the antiviral activity of Echinaforce against these viruses and found that the antiviral effects against MERS-CoV (Fig. 4a) and SARS-CoV (Fig. 4b) were comparable with the effect observed for HCoV-229E. Interestingly, MERS-CoV was even more sensitive than HCoV-229E to pre-treatment with the lower concentration (10 µg/ml) of Echinaforce.

Echinaforce reduces infectivity other membranous viruses in vitro
Similar antiviral activity was observed for yellow fever virus, another enveloped RNA virus (Fig. 5a). In contrast, Echinaforce showed no effect at all on the infectivity of vaccinia virus (Fig. 5b) and the minute virus of mice (Fig. 5c), which are DNA viruses, with and without an envelope, respectively.

Discussion
Broadly Herbal preparations of Echinacea have traditionally been used to prevent and treat symptoms of colds and flu and are still widely used (9,12). Echinaforce, an Echinacea purpurea extract, has been shown to broadly inhibit the infectivity of influenza A and B, RSV, parainfluenza virus, and herpes simplex virus in-vitro and to interfere with cytokine production induced upon viral infection (19)(20)(21). Results from the current study complement these previous findings by demonstrating a direct antiviral activity of Echinaforce both against common cold coronavirus 229E (HCoV-229E) and highly pathogenic coronaviruses (SARS-CoV and MERS-CoV). We observed a dose dependent inactivation of HCoV-229E upon direct exposure to the extract and 50% reduction of HCoV-229E infectivity (IC 50 ) was achieved at 3.2 µg/ml. As previously seen for RSV, limited Mechanism of action of different Echinacea extracts are currently unclear, however, for most viruses, Echinaforce seems to exert its antiviral effect upon direct contact, leading to a permanent inactivation of the virus particles. In the current study, inhibition of HCoV-229E infectivity after direct exposure could not be reverted by washing. This observed effect is likely due to a stable alteration of viral components, presumably, the viral membrane, or membrane proteins. Although specific inhibition has been suggested for Influenza (19), the heterogeneity of the envelope proteins and cell receptors used by all the different viruses susceptible to Echinacea treatment strongly argues against a specific mechanism of action. Rather, the broad antiviral activity of Echinacea on various membranous RNA viruses points to a more general inhibitory effect. Non-enveloped rhinoviruses are sensitive to Echinaforce at high concentrations while adenoviruses and mouse parvovirus are not (20). Interestingly, Echinacea does not inhibit vaccinia virus, a large, enveloped DNA virus. So far, it is the only enveloped virus found to be resistant to treatment with Echinaforce.
We investigated whether a protective effect in the upper-respiratory tract could be reproduced in-vitro, in re-constituted three-dimensional nasal epithelium, i.e air-liquid interface (ALI) cell cultures, where the apical side is exposed to air resembling the human airways in-vivo. This cell culture system recapitulates many of the characteristics of the human respiratory tract, including ciliary beating and mucus production (27,28). Regular These studies indicate a clinically relevant protection against coronaviruses with prophylactic Echinaforce treatment at tolerable and safe dosages. Furthermore, we have also observed partial protection at lower concentrations. In vivo, this might be due to insufficient dosage. A better protection may be achieved by ingesting higher doses of the extract or a more directed distribution of Echinaforce in the airways, e.g. by aerosol delivery. Furthermore, isolation and concentration of the active compounds in Echinacea products could result in smaller daily doses and increased activity.
As previously mentioned, in addition to direct inactivation of viral particles, Echinacea also inhibits cytokine secretions during virus infection. Excessive production of interleukin-6 (IL-6) or IL-8 have been associated with symptomatic development of viral infections and such responses, i.e. a cytokine storm, are likely responsible for many of cold-associated symptoms such as runny nose, coughing, sneezing et cetera (29). During certain viral infections (e.g. influenza), the heightened immune response may actually contribute to the destruction of respiratory epithelium and may even be the dominant reason for symptoms in absence of virus-mediated cytopathicity (30,31). In these cases, the inhibition of virus-induced cytokine production by Echinaforce may be beneficial by limiting the damage of the respiratory epithelium provoked by the immune system (13).
For many other viruses, including coronaviruses, no direct cell destruction is observed during infection. This is in accordance with the fact that coronaviruses, in general, do not elicit a pronounced cytokine response upon infection (32). Despite severe symptoms and pulmonary pathology, the highly pathogenic MERS-CoV does not elicit an overwhelming cytokine response in primary respiratory epithelial cells in the early course of infection.
However, later on, a marked induction of the pro-inflammatory cytokines/chemokines IL-1β, IL-8 and IL-6 was observed (33). Even if the anti-inflammatory action of Echinaforce is less relevant for coronaviruses, treatment with 50µg/ml Echinaforce inactivated both MERS-CoV and SARS-CoV particles to similar levels as observed for HCoV-229E.

Conclusions
In the current study, we have shown that human coronaviruses are readily inhibited by Echinaforce in vitro, further strengthening its use as a prophylactic treatment against a wide range of respiratory viruses causing either serious pulmonary disease or the common cold. Furthermore, a broadly acting antiviral compound suitable for long-term prophylaxis upon exposure could potentially reduce the high mortality rates associated with MERSand SARS-CoV infections. Due to its general mode of action, novel zoonotic coronaviruses, such as SARS-CoV-2, could also be sensitive to Echinaforce, potentially providing an accessible and inexpensive prophylactic treatment for emerging coronavirus infections.    (a) MERS-CoV is highly sensitive to direct Echinaforce treatment, with significant reduction in viral titer observed at 10µg/ml and complete inactivation at 50µg/ml.
(b) SARS-CoV is completely inactivated at the highest concentration with a slight reduction in viral titer after exposure to 10µg/ml. No effect was observed on cell viability (right y-axis). The data shown are representative of two independent experiments (mean ± sd).