J Bacteriol Virol.  2019 Dec;49(4):203-211. 10.4167/jbv.2019.49.4.203.

Antiviral Activity of Gemcitabine Against Echovirus 30 Infection in Vitro

• Choi HJ
• Song JH
• Yoon K

• KMID: 2468026
• DOI: http://doi.org/10.4167/jbv.2019.49.4.203

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.

Keyword

Echovirus 30; Gemcitabine; Early stages

MeSH Terms

Adult
Antiviral Agents
Child
Enterovirus B, Human*
Humans
In Vitro Techniques*
Life Cycle Stages
Meningitis
RNA, Viral
Antiviral Agents
RNA, Viral

Figure

• 

  Figure 1 Gemcitabine-mediated antiviral activity against E30 in vitro. (A) RD cells were infected with the TCID50 (50 % cell culture infective dose) of E30, and treated with indicated concentrations of gemcitabine and rupintrivir. Cell viability was evaluated using an SRB assay, and results were determined based on absorbance at 524nm. Bar graphs show mean ± SD. Control (Ctrl) was neither infected nor treated, while vehicle (Veh) was infected with E30 but not treated with gemcitabine and rupintrivir. (B) The cytotoxicity of gemcitabine was evaluated using an SRB assay in RD cells. ***P<0.001, based on ANOVA with Bonferroni's multiple comparison test.

• 

  Figure 2 Time course of E30 infection. RD cells infected with the TCID50 of E30 were harvested at the indicated time points after addition of 10 µM gemcitabine, or 2 µM rupintrivir (post-infection). Total RNA was isolated and E30 RNA was analyzed using RT-qPCR. Real-time PCR analyses were performed to determine the effect of gemcitabine, or rupintrivir on E30 NCR gene expression levels. Each value is the result of mean ± S.D. of three independent experiments.

• 

  Figure 3 Time-of-addition experiment to test effect of gemcitabine on the E30 viral cycle. Gemcitabine (10 µM), or rupintrivir (2 µM) were added prior to, at the time of, or at indicated time points after viral infection of RD cells. The level of viral RNA expression was analyzed 14h post-infection. RD cells that were treated with drugs prior to viral infection were washed before infection. Each value is the result of mean ± S.D. of three independent experiments.

• 

  Figure 4 Effects of gemcitabine on the infectivity of E30 particles. E30 particles were incubated with 10 µM gemcitabine or 2 µM rupintrivir for 1h at 4℃. RD cells were then incubated with the treated or untreated virus for 1 h at 37℃. Unbound virus was removed by extensive washing, and incubation was continued with or without 10 µM gemcitabine or 2 µM rupintrivir, at 37℃. Antiviral activity was determined using RT-qPCR, 12 h post-infection. Preinc, pre-incubation of virus with the indicated drug without subsequent drug treatment of the infected cells; Inc., incubation of cells with the indicated drug after viral infection.

• 

  Figure 5 NTP inhibits the antiviral activity of gemcitabine in vitro. E30-infected RD cells were incubated with gemcitabine for 4 8 h at the indicated concentrations and (A) ATP, (B) GTP, (C) CTP, (D) UTP, and cell viability was determined. Data is expres sed as mean ± SD of the cell viability percentage values obtained from three independent experiments carried out in a triplic ate. ***P<0.001 for comparisons to the control, and #P<0.1, ##P<0.01, ###P<0.001 for comparisons between NTP treatment and NTP non-treatment, and at the corresponding dose of gemcitabine.

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