With respect to RNA viruses, EGCG was tested against two other viruses, enterovirus 71 belonging to the family of Picornaviridae and influenza viruses, which are members of the family of Orthomyxoviridae.
Influenza A and B viruses are a major cause of respiratory disease in humans. In addition, influenza A viruses continuously re-emerge from animal reservoirs into humans causing human pandemics of unpredictable severity every 10–50 years (Garcia-Sastre, 2011). Influenza A viruses are negative sense, single-stranded, segmented RNA viruses with an envelope. There are several subtypes known, labelled according to an H number (for the type of haemagglutinin) and an N number (for the type of neuraminidase). The annual flu (also called ‘seasonal flu’ or ‘human flu’) results in approximately 36 000 deaths and more than 200 000 hospitalizations each year in the USA alone. Vaccines are the most widely used intervention prophylaxis for influenza infection, but their effectiveness depends on the type of influenza virus and they also have the drawback of limited supply (Collin and de Radigues, 2009). Two main classes of antiviral drugs used against influenza viruses are neuraminidase inhibitors or inhibitors of the viral M2 protein, such as amantadine and rimantadine. These drugs can reduce the severity of symptoms and mortality and can also be taken to decrease the risk of infection. However, viral strains have emerged that show drug resistance to both classes of drug. The antiviral activity of EGCG against influenza virus was reported for the first time in 1993. The green tea molecule affected the infectivity of influenza virus in cell culture and it was shown to agglutinate the viruses, preventing the virus from absorbing to MDCK cells (Nakayama et al., 1993). Furthermore, green tea extracts including EGCG exerted an inhibitory effect on the acidification of intracellular compartments such as endosomes and lysosomes, resulting in inhibition of influenza virus growth in tissue culture (Imanishi et al., 2002). These studies were extended by Song et al.; they determined the structure–activity relationship of the different green tea polyphenolic compounds EGCG, ECG and EGC against influenza and found that ECG and EGCG were more effective than EGC and these molecules also exerted an inhibitory effect on the neuraminidase in a biochemical assay (Song et al., 2005). Influenza viral RNA synthesis analysed by RT-PCR was affected only at very high concentrations (Song et al., 2005). Interestingly, based on these in vitro data clinical studies were performed to investigate if green tea catechins can prevent influenza infections in humans. In a small prospective cohort study it was reported that gargling with tea catechins extracts was effective in preventing influenza infection in elderly residents of a nursing home (Yamada et al., 2006). In addition, in another randomized, double-blind, placebo-controlled trial, the consumption of catechins for 5 months had a statistically significant preventive effect on clinically defined influenza infection and was well tolerated (Matsumoto et al., 2012). The results of these trials indicate these catechins have a protective effect against influenza virus; however, large-scale studies are needed to confirm this.
Enterovirus 71 is a single-stranded RNA virus and one of the causative agents of hand, foot and mouth disease (HFMD). This virus causes various clinical manifestations, including cutaneous, visceral and neurological diseases. Large outbreaks have been reported in Taiwan and Malaysia in the 1990s. Recently, enterovirus 71 repeatedly caused life-threatening outbreaks of HFMD with neurological complications in Asian children. The neurological manifestations progress very quickly and range from aseptic meningitis to acute flaccid paralysis and brainstem encephalitis. EGCG was demonstrated to inhibit enterovirus 71 replication and formation of infectious progeny virus (Ho et al., 2009). There was a positive correlation between the antioxidant effects of catechins (Yang et al., 1994) and their antiviral activity (Ho et al., 2009). These findings suggest that EGCG may suppress viral replication via modulation of the cellular redox milieu.
The aetiological agent of acute and chronic hepatitis B is human hepatitis B virus (HBV), a small enveloped virus from the family of Hepadnaviridae. Around 40% of the global human population has contact with this virus, which is transmitted parentally, sexually and perinatally (Shepard et al., 2006). Infection results in acute hepatitis and – in some cases – acute liver failure. Chronic hepatitis B, which affects over 300 million, persists even after clinical resolution of acute infection and can be reactivated causing severe disease under conditions of immunosuppression. In contrast to HCV, a preventive vaccine for HBV and specific antiviral drugs are available. However, viral resistance increasingly poses a challenge (Tillmann, 2007). To elucidate the effect of green tea catechins on HBV, the influence of green tea extracts and EGCG was studied in a stable cell line, HepG2-N10, expressing HBV antigens. It was found that the expression of HBV-specific antigens, the levels of extracellular HBV DNA, intracellular replicative intermediates and cccDNA were reduced in a dose-dependent manner (Xu et al., 2008). However, it is difficult to dissect the detailed anti-HBV mechanisms of EGCG using HepG2-N10 cells as the process from cccDNA to antigen expression are strongly affected by transcription of integrated HBV DNA (Zhou et al., 2006). Therefore, more recently, He et al. (2011) used an inducible HBV replicating cell line to test EGCG, termed HepG2.117, where HBV precore mRNA can only be transcribed from replicating HBV DNA, but not the integrated HBV DNA. They observed that HBV replicative intermediates of RNA synthesis were significantly inhibited by EGCG, which resulted in a reduction in cccDNA production (He et al., 2011). In contrast, the production of HBV pregenomic RNA, precore mRNA and the translation of hepatitis B e antigen (HBVeAg) were not affected. To elucidate whether the antiviral effect of EGCG is the result of targeting of cellular factors or viral factors, additional studies are required, ideally in cell culture models that replicate the complete HBV life cycle.
In the case of other DNA viruses, so far the effects of EGCG against adenovirus, Epstein–Barr virus (EBV) and HSV-1 have been studied, the two latter viruses belong to the family of Herpesviridae. Adenoviruses are non-enveloped viruses composed of a nucleocapsid and a double-stranded linear DNA genome. There are 57 serotypes described in humans, which are responsible for 5–10% of upper respiratory infections in children. Humans infected with adenoviruses display a wide range of responses, from no symptoms at all to the severe infections typical of adenovirus serotype 14. Green tea was found to reduce the virus yield of an adenovirus infection by two orders of magnitude in Hep2 cells (Weber et al., 2003). The molecule was most effective when added to the cells during the transition from early to late phase of viral infection suggesting EGCG inhibits one or more late steps in the viral infection (Weber et al., 2003). Furthermore, inactivation of purified adenoviruses and inhibition of viral protease activity was noted. However, its therapeutic value seems to be limited, as the effective concentrations were much higher than reported serum concentrations of green tea drinkers. This was also the case when EGCG was tested against EBV. EBV is a human herpesvirus causing infectious mononucleosis and is closely associated with Burkitt's lymphoma, nasopharyngeal carcinoma, T-cell lymphoma and Hodgkin's disease (Bravender, 2010). In vitro, only an EGCG concentration exceeding 50 μM decreased the expression of EBV lytic proteins, including Rta, Zta and EA-D, but not the expression of EBV nuclear antigen-1 (Chang et al., 2003). Moreover, DNA microarray and transient transfection analysis demonstrated that this concentration of EGCG blocked the EBV lytic cycle by inhibiting the transcription of immediate-early genes (Chang et al., 2003).
Herpes simplex is a viral disease caused by HSV-1 and type 2 (HSV-2). Worldwide rates of HSV infection are between 65 and 90% (Chayavichitsilp et al., 2009). There is no vaccine available or a method to eradicate herpes viruses from the body, but antiviral medications like acyclovir can reduce the frequency, duration and severity of outbreaks. Characterization of the antiviral activity of EGCG against HSV-1 and HSV-2 revealed that EGCG has greater anti-HSV activity than other green tea catechins and inactivated multiple clinical isolates of HSV-1 and HSV-2. Importantly, EGCG reduced HSV-2 titres by more than 1000-fold in 10–20 min and reduced HSV-1 titres to the same extent in 30–40 min (Isaacs et al., 2008). Similar to HCV, HIV-1 and influenza virus, the anti-HSV activity was due to a direct effect on the virion and incubation of target cells prior to infection had no effect (Isaacs et al., 2008). Using electron microscopy, the authors showed that purified viruses exposed to EGCG were damaged. As EGCG is stable in the pH range found in the vagina, it was proposed that the green tea molecule could be a promising candidate for use in a microbiocide to reduce HSV transmission (Isaacs et al., 2008). Furthermore, EGCG dimers inactivated HSV-1 and HSV-2 more effectively between pHs 4.0 and 6.6 than the EGCG monomer, which has therefore even more potential for reducing spread of HSV in vivo (Isaacs et al., 2011).