EV71 belongs to enterovirus genus of the Picornaviridae family, and is one of the main pathogens causing hand, foot and mouth disease in infants. The development process of antiviral drugs is slow, coupled with the special population of infants and young children, so there is no specific drug to treat the virus infection[20, 21]. Taken together, it is of great significance to develop safe and effective drugs against EV71 infection.
Ellagic acid, a natural polyphenolic compound that is widely distributed in fruits and nuts, has a variety of biological functions. But its low absorbtion efficiency restricts the application as a drug. Urolithins are its metabolites, which also have a variety of biological functions, and can achieve ideal blood drug concentration after absorption through the intestinal tract[7, 17]. In this study, we screened UroA, UroB and UroC for antiviral effects. The results showed that UroA has a significant antiviral effect, which was fully confirmed from CPE, the viral protein expression, the copy of viral RNA and the virus titers. The cytotoxicity and anti-EV71 activities of UroA was also investigated from CC50, IC50 and selection index (SI). The results showed that compared with ribavirin as positive control, UroA has better safety and antiviral effects, which has potential application value and can be used as a good candidate drug. In this study, the functions of UroA in the prevention, treatment and direct viricidal effects of EV71 were also tested, respectively. The results showed that UroA has a good function in the treatment of EV71 infection, but little effect of the prevention and no direct viricidal effect of EV71.
Autophagy and apoptosis induced by virus have both positive and negative effects on the proliferation on the virus[3, 25]. In the late stage of EV71 infection, cell autophagy and apoptosis can be promoted, which is conducive to the maturation and release of progeny virions and promote the proliferation of viruses. However, in the early stage of EV71 infection, cells recognize virions and quickly initiate autophagy and apoptosis mechanisms to prevent the generation of progeny virions and inhibit the proliferation of the virus. In this study, we examined the effects of UroA on autophagy and apoptosis of EV71 infected cells. The results showed that UroA promoted cell autophagy and apoptosis in the early stage of EV71 infection, which facilitates virus clearance. This is also the reason why autophagy and apoptosis of cells in the UroA group were significantly less than those in the control group in the following time.
Some clinical case reports have shown that EV71 infection can induce nerve damage, and the replication of EV71 in nerve cells has also been reported[10–12, 27]. In this study, the effects of UroA on the proliferation of EV71 in SK-N-SH cells was also detected. The results showed that UroA could significantly inhibit the proliferation of EV71 and the antiviral effects of UroA were also superior to ribavirin in SK-N-SH cells.
In this study, the effects of UroA against influenza virus were also tested. Western-blot assay showed that UroA can not inhibit influenza virus replication. This suggests that the antiviral effects of UorA are not broad-spectrum. However, the effects of UroA on the proliferation of other viruses still need to be verified.
In conclusion, our study demonstrated that UroA exerted more potent-antiviral activity against EV71 in vitro and has better safety compared with ribavirin. The anti-EV71 mechanism of UroA may be related with promoting autophagy and apoptosis of infected cells. Therefore, UroA can be used as a potential drug candidate and is worthy of further study.