Ribavirin inhibits the replication of infectious bursal disease virus predominantly through depletion of cellular guanosine pool

Abstract

Introduction: The antiviral activity of different mutagens against single-stranded RNA viruses is well documented; however, their activity on the replication of double-stranded RNA viruses remains unexplored. This study aims to investigate the effect of different antivirals on the replication of a chicken embryo fibroblast-adapted Infectious Bursal Disease virus, FVSKG2. This study further explores the antiviral mechanism utilized by the most effective anti-IBDV agent.

Methods: The cytotoxicity and anti-FVSKG2 activity of different antiviral agents (ribavirin, 5-fluorouracil, 5-azacytidine, and amiloride) were evaluated. The virus was serially passaged in chicken embryo fibroblasts 11 times at sub-cytotoxic concentrations of ribavirin, 5-fluorouracil or amiloride. Further, the possible mutagenic and non-mutagenic mechanisms utilized by the most effective anti-FVSKG2 agent were explored.

Results and discussion: Ribavirin was the least cytotoxic on chicken embryo fibroblasts, followed by 5-fluorouracil, amiloride and 5-azacytidine. Ribavirin inhibited the replication of FVSKG2 in chicken embryo fibroblasts significantly at concentrations as low as 0.05 mM. The extinction of FVSKG2 was achieved during serial passage of the virus in chicken embryo fibroblasts at ≥0.05 mM ribavirin; however, the emergence of a mutagen-resistant virus was not observed until the eleventh passage. Further, no mutation was observed in 1,898 nucleotides of the FVSKG2 following its five passages in chicken embryo fibroblasts in the presence of 0.025 mM ribavirin. Ribavarin inhibited the FVSKG2 replication in chicken embryo fibroblasts primarily through IMPDH-mediated depletion of the Guanosine Triphosphate pool of cells. However, other mechanisms like ribavirin-mediated cytokine induction or possible inhibition of viral RNA-dependent RNA polymerase through its interaction with the enzyme's active sites enhance the anti-IBDV effect. Ribavirin inhibits ds- RNA viruses, likely through IMPDH inhibition and not mutagenesis. The inhibitory effect may, however, be augmented by other non-mutagenic mechanisms, like induction of antiviral cytokines in chicken embryo fibroblasts or interaction of ribavirin with the active sites of RNA-dependent RNA polymerase of the virus.

Keywords: antiviral; dsRNA; infectious bursal disease virus; mutagen; ribavirin.

Figure 1

Cytotoxicity assay of mutagens on CEFs: Cell viability was determined by using MTT assay: Y-axis represents percent cell viability, while X-axis represents hours post-treatment (hpt). Data expressed as mean ± standard error (n = 3). Significant cytotoxicity was observed at and above 1.0 mM of ribavirin, while as in case of 5-fluorouracil, 5-azacytidine and amiloride significant cytotoxicity was observed at 0.5 mM. Asterisks represent significant difference (p < 0.05). The number of asterisks “*” represents the extent of significance. The 0 hpt refers to time period immediately after the treatment.

Figure 2

Effect of ribavirin, 5- fluorouracil, 5-azacytidine, and amiloride on replication of FVSKG2: The effect of indicated concentrations of the mutagens on the replication of IBDV isolate FVSKG2 in CEFs were evaluated. Results are presented as 50% tissue culture infective dose TCID50 per mL (y-axis) over time (x-axis). Virus titers were evaluated using cell culture fluids collected from pre-treated CEFs every 24 h after being incubated with the FVSKG2. At 0.05, 0.1, 0.2, and 0.3 mM of ribavirin, the replication of FVSKG2 significantly decreased in a dose-dependent manner. At 0.5 mM of 5-fluorouracil, a 2 log₁₀ drop in virus titer was seen along with significant suppression of FVSKG2 replication (p = 0.0001). On the other hand, there was no detectable antiviral action with 0.1, 0.2, and 0.3 mM 5-fluorouracil. Treatment with 5-azacytidine at 0.2 and 0.3 mM significantly reduced FVSKG2 replication by up to 2 log₁₀ (p = 0.0019). Similarly, significant inhibition of FVSKG2 replication was caused by high concentrations (1 and 2 mM) of amiloride. Error bars denote mean ± standard error of the mean (SEM) while Asterisks “*” indicate significant differences in virus titer (TCID50/mL log₁₀) as compared to control (0 mM), p < 0.05. The number of asterisks represents the extent of significance.

Figure 3

Sequential passage of FVSKG2 in different concentrations of ribavirin, 5-fluorouracil, and amiloride in CEFs: In presence of ribavirin at concentrations of 0.05 and above 0.1 mM, extinction of virus occurred during third and first passage, respectively. In presence of 5-fluorouracil no significant inhibition of FVSKG2 was observed at concentrations at and above 0.1 mM in CEFs. In presence of amiloride, no significant inhibition of FVSKG2 was observed at concentration at and above 0.05 mM in CEFs, Asterisks “*” indicate significant differences.

Figure 4

Ribavirin competition assay with guanosine: Ribavirin (Rib) competition assay with nucleoside guanosine (Guo) was performed with FVSKG2 at MOI of 0.01 in CEFs, under three different conditions: no drug treatment, 0.05 mM of ribavirin and 0.05 mM ribavirin plus 0.025 mM guanosine. Culture supernatants were harvested at 48 h post-treatment to determine viral titers. The mean viral titers ± s.d. from triplicates derived from one out of two independent experiments are shown.

Figure 5

(A) The effect of indicated concentrations of ribavirin (Rib) and mycophenolic acid (MPA) in presence or absence of Guanosine (Guo) Supplementation on the replication of FVSKG2 in CEFs. Virus titers are presented as 50% tissue culture infective dose (TCID50/mL, log₁₀) per milliliter (y axis) over drug concentration (x axis). Errors bars denote mean ± standard error of mean (SEM). (B) B-factor diagram indicating binding free-energy contribution of interacting residues. (C) FEL values constructed as a function of PC1 and PC2 eigenvectors. (D) The ligand-IMPDH interaction diagram showing the presence of important interactions.

Figure 6

Evaluation of cytokines alteration in CEF cells by ribavirin: CEFs were grown in the presence or absence of indicated concentrations of ribavirin. CEFs were harvested at 24 hpt and subjected to cytokine mRNA expression analysis. Cytokines expression analysis of IL-6, IL-2, IL-12, interferon-alpha (IFN-α), tumor necrosis factor-alpha (TNF-α) and TNF-β mRNAs in CEF cells after 24-h culture. qRT-PCR was used for evaluating mRNA expression. While Relative quantification (RQ) was calculated using the 2-DDCt method. Error bars denote mean ± standard error of the mean (SEM). The bars represent the means, and the error bars represent the standard errors of the mean (SEM). Bars showing different letters represent values that differ significantly from each other (p < 0.05).

Figure 7

Interaction of ribavirin with RdRp: (A) Denotion of the target sites in the IBDV RdRp structure. (B) Autodock vina based Molecular docking analysis of ribavirin at site 1 and site 2. (C) Analysis of interacting residues and nature of interaction between ribavirin and IBDV RdRp target sites.