An Investigation of the Effect of Transfected Defective, Ebola Virus Genomes on Ebola Replication

Abstract

As the ongoing outbreak in the Democratic Republic of Congo illustrates, Ebola virus disease continues to pose a significant risk to humankind and this necessitates the continued development of therapeutic options. One option that warrants evaluation is that of defective genomes; these can potentially parasitize resources from the wild-type virus and may even be packaged for repeated co-infection cycles. Deletion and copy-back defective genomes have been identified and reported in the literature. As a crude, mixed preparation these were found to have limiting effects on cytopathology. Here we have used synthetic virology to clone and manufacture two deletion defective genomes. These genomes were tested with Ebola virus using in vitro cell culture and shown to inhibit viral replication; however, and against expectations, the defective genomes were not released in biologically significant numbers. We propose that EBOV might have yet unknown mechanisms to prevent parasitisation by defective interfering particles beyond the known mechanism that prevents sequential infection of the same cell. Understanding this mechanism would be necessary in any development of a defective interfering particle-based therapy.

Keywords: DIPs; defective interfering particles; deletion; ebola; in vitro.

Figure 1

Genetic maps of the defective genome constructs from Calain et al. (1999). The Light orange region correspond to regions of the backbone plasmid (pUC 57_A338); the gray regions correspond to the virus regions that were used from the EBOV viral sequence (KJ660347.2). The colors are for guidance only.

Figure 2

The effect of defective genomes on viral release. 2 × 10⁵ Vero c1008 cells were infected with 1 × 10⁶ TCID₅₀ infectious units of EBOV strain Ecran (~1 × 10¹² genomes) for 2 h then washed. The cells were then transfected with 1 μg of control RNA (red dots) or DG-d1 RNA (blue dots) RNA or DG-d2 RNA (purple dots) for 4 h. Un-treated Ebola virus only infection was used for comparison. The cells were then incubated and cell culture supernatants at 48 h post infection were taken and analyzed. The virus was enumerated using qRT-PCR (A) and TCID50 assay (B). Data shown are normalized to the EBOV-Ecran only control for each experiment. One sample T-tests were performed to indicate the likelihood that the difference would have occurred through random chance. The P-values are given for the 95% level. Each data point is an independent experiment and the geometric mean of 3 replicates generated from independent wells within the same experiment.

Figure 3

The release of defective genomes. 2 × 10⁵ Vero c1008 cells were infected with 1 × 10⁶ TCID₅₀ infectious units of EBOV strain Ecran (~1 × 10¹² genomes) for 2 h then washed. The cells were then transfected with 1 μg of DG-d1 RNA for 4 h. The cells were then incubated. Samples of the cells at time 0 and supernatant at 48 h post infection were taken. The viral genomes (defective and wild-type) were enumerated using qRT-PCR. The graph shows the count of each viral type (wild-type and DG-d1) within the cells at the onset of the experiment and the counts of viral type in the extracellular space after 48 h. Each data point is the geometric mean (± 95% confidence intervals) of 3 independent experiments, where each experiment is the geometric mean of 3 replicates generated from independent wells within the same experiment. Where EBOV Ecran was measured, black lines link the data points and the gray circles indicate non-transfected control infected cells, the red circles indicate infected cells transfected with control RNA and the blue circles indicate the infected cells transfected with DG-d1. Where the DG-d1 was measured, the green lines link the data points and the green circles indicate the infected cells transfected with DG-d1 and the yellow circles indicate uninfected cells transfected with DG-d1.