Antiviral Activity of Gemcitabine Against Echovirus 30 Infectionin Vitro

Abstract

Echovirus 30 is one of the major causes of meningitis in children and adults. The purpose of our current study was to investigate whether selected antiviral drugs could provide antiviral activity against echovirus 30. Using RD cells, we assessed the cytopathic effect of echovirus 30, including viral RNA levels as indicators of viral replication. The effects of gemcitabine were compared to rupintrivir, a well-known antiviral drug. To understand the activity gemcitabine exerts on the viral life cycle, time course and time-of-addition assays were implemented. The most effective compounds against echovirus 30 were gemcitabine and rupintrivir, as demonstrated by their concentration-dependent activity. Gemcitabine affects the early stages of echovirus 30 infection by disrupting viral replication. However, gemcitabine failed to directly inactivate echovirus 30 particles or impede viral uptake into the RD cells. Gemcitabine can be considered as a lead candidate in the development of echovirus 30 antiviral drugs, specifically in the early stages of echovirus 30 replication. Based on the results produced from the E30 in vitro assay, we conducted time course experiments to analyze the mechanism of action of gemcitabine. Viral RNA from E30 was detected using RT-PCR at various times points (4, 6, 8, 10, and 12 h post-infection) and after gemcitabine (10  M) and rupintrivir (2  M) treatment of E30-infected cells. We could find E30 viral RNA as early as 8 h post-infection. Interestingly, gemcitabine was seen to block the proliferation of E30 in resemblance to rupintrivir (inhibitor of picornavirus 3C protease, Fig. 2). Based on the results of the time course experiments, we hypothesized that the antiviral effect of gemcitabine occurs relatively early in the infection process. To observe which step is influenced by gemcitabine, we performed a time-of-addition experiment. Gemcitabine (10  M) and rupintrivir (2  M) were added to the culture medium at -1, 0, 1, 2, 4, 6, 8, 10 and 12 h after virus infection. E30 RNA replication was analyzed infection. Therefore, we concluded that the antiviral effect of gemcitabine occurs relatively early during viral infection. To realize the potential of gemcitabine as an antiviral candidate, its mechanism of activity must be understood. It is important to differentiate between viral inactivation (virucidal activity) from antiviral activity. Direct viral inactivation is an early event where the virus is inactivated before it infects the cells while antiviral activity involves killing the virus or the suppression of viral replication. It would be ideal for viral infection treatments to possess both virucidal and antiviral activities. In our study, gemcitabine did not directly interact with E30 particles as pre-exposure of the virus to gemcitabine did not alter the infectivity of E30 particles. Furthermore, addition of UTP and CTP significantly reduced the antiviral activity of gemcitabine by increasing the levels of pyrimidine nucleotides. The anti-echoviral activity of gemcitabine may be altered by reducing the levels of CTP and UTP after having being converted into gemcitabine triphosphate. Thus, this study has revealed that gemcitabine could possess antiviral activity against E30. It does not directly inactivate E30 particles, but affects the initial stages of E30 infection by interfering with viral replication. The antiviral activity induced by gemcitabine treatment may be adjusted by reducing the levels of CTP and UTP after it has been converted into gemcitabine triphosphate. These results could be useful in the design of new inhibitors for use as anti-viral agents.