Inhibition of enterovirus 71 replication by chrysosplenetin and penduletin

Abstract

In recent years, enterovirus 71 (EV71) infections have caused an increasing epidemic in young children, accompanying with more severe nervous system disease and more deaths. Unfortunately, there is no specific medication for it so far. Here we investigated the anti-EV71 activity of chrysosplenetin and penduletin, two o-methylated flavonols isolated from the leaves of Laggera pterodonta. These two compounds were found to have strong activity in vitro against EV71 with low cytotoxicity. In the cytopathic effect (CPE) inhibition assays, both plaque reduction assay and virus yield inhibition assay, the compounds showed a similar 50% inhibitory concentration (IC₅₀) value of about 0.20 μM. The selectivity indices (SI) of chrysosplenetin and penduletin were 107.5 and 655.6 in African green monkey kidney (Vero) cells, and 69.5 and 200.5 in human rhabdomyosarcoma (RD) cells, accordingly. The preliminary mechanism analysis indicates that they function not through blocking virus entry or inactivating virus directly but inhibiting viral RNA replication. In the time-of-addition assay, both compounds inhibited progeny virus production and RNA replication by nearly 100% when introduced within 4 h post infection. In addition to EV71, both compounds inhibited several other human enteroviruses with similar efficacy. These findings provide a significant lead for the discovery of anti-EV71 drug.

Introduction

Human enteroviruses belong to the Enterovirus genus of the Picornaviridae family and comprise a major subgroup of small RNA(+) viruses (Palacios and Oberste, 2005), which includes polioviruses, coxsackieviruses, echoviruses, and enteroviruses. Most of these viruses establish infection via the intestinal epithelial tract and lymphoid tissues, and cause paralytic disease, aseptic meningitis, exanthems, myocarditis, pericarditis, and nonspecific febrile illness. Enterovirus 71 (EV71) infections frequently manifest as hand, foot, and mouth disease, as well as encephalitis in infants and young children. EV71 can even cause severe central nervous system disease, complications, and fatalities (Huang et al., 1999, Lee et al., 2009, Solomon et al., 2010). Since it was first reported in 1974 (Schmidt et al., 1974), EV71 has been implicated in several outbreaks worldwide, especially in the Asia–Pacific region (Ooi et al., 2010, Weng et al., 2010). According to the latest reports from the Chinese Ministry of Health, there were over 1 million reported cases of EV71 infection in China in 2010, including 15,000 severe cases and more than 600 fatalities (Ministry of Health of the People’s Republic of China, 2010).

At present, there is no specific treatment available for EV71 (Ooi et al., 2010). In the face of frequent outbreaks and the fatal consequences of EV71 infection, antiviral drugs for EV71 are urgently needed. Previous studies indicate that ribavirin and Type I interferon reduces mortality in EV71-infected mice (Liu et al., 2005, Li et al., 2008). Pleconaril, a Sterling-Winthrop (WIN) compound targeting viral protein 1 (VP1), is a successful clinical candidate for most enteroviruses, but not EV71 (Pevear et al., 1999, Shia et al., 2002, Florea et al., 2003, De Palma et al., 2008). On the other hand, pyridyl imidazolidinones derived from WIN compound templates show promising anti-EV71 activity in tissue cultures (Shia et al., 2002, Chen et al., 2009).

Laggera pterodonta has been used as traditional medicine in China for several centuries for its anti-inflammatory and antibacterial properties (Jiangsu New Medical College, 1977). L. pterodonta belongs to Compositae and is widely distributed in Southwestern China, especially in Yunnan and Sichuan Provinces. A number of chemical constituents have been isolated from this plant, mainly flavones and triterpenes (Liu et al., 2010). Chrysosplenetin and penduletin are two known flavonols that have been isolated from the leaves of L. pterodonta (Li and Ding, 1994). Besides penduletin has been reported to have anti-poliovirus activity and anti-inflammatory activity (De Meyer et al., 1991, Moscatelli et al., 2006), very little is known of the medicinal properties of these two flavonols.

In this study, the anti-EV71 activity of chrysosplenetin and penduletin were determined in African green monkey kidney (Vero) cells and human rhabdomyosarcoma (RD) cells using an EV71 infection-induced cytopathic effect (CPE) assay and a plaque reduction assay. And their modes of action were also prelimitarily determined. In addition to the anti-EV71 studies, the antiviral activity of these two compounds against other enteroviruses was investigated.