Inhibition of Japanese encephalitis virus replication in cultured cells and mice by a peptide-conjugated morpholino oligomer

Abstract

Background: Japanese encephalitis virus (JEV) has a significant impact on public health throughout Asia, and there is a pressing need for development of new therapeutics against it.

Methods: Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) are antisense agents that enter cells readily and interfere with gene expression. Four PPMOs, targeting various locations in the JEV genome, were evaluated for antiviral activity against JEV in cultured cells and the mouse model of JEV infection.

Results: A PPMO (P10882) targeting the JEV 3' cyclization sequence (3'CSI) had significant antiviral activity in Vero (epithelial), Neuro2A (neuronal) and J774E (macrophage) cells at concentrations that were not cytotoxic. P10882 added before infection suppressed JEV replication to an undetectable level in Vero cells and produced a 93% and 66% reduction in titre in J774E and Neuro2A cells, respectively, when measured at 24 h post-infection. In uninfected cells, fluorescein-labelled PPMOs entered J774E cells most efficiently, followed by Vero and Neuro2A cells. The antiviral effect of P10882 was also demonstrated in vivo, where 60%-80% of 1-week-old mice treated intracerebrally with a 20 mg/kg dose of P10882 every 12 h for 5 days were protected from a lethal dose of JEV and showed an undetectable level of virus in brain tissue at 2 days post-infection.

Conclusions: P10882, which targets sequence that is highly conserved across members of the JEV serocomplex, was previously shown to be effective in a mouse model of West Nile disease, and represents a candidate antiviral agent against members of the JEV serocomplex.

Figure 1

PPMO target locations in the JEV genome, and sequence conservation at the 3′CSI region. (a) Diagram showing the secondary structure of 5′- and 3′-UTRs of JEV genomic RNA, as predicted by the Mfold program. The PPMO target sequences are indicated with names, lines and arrows. The nucleotides of the AUG translation initiation codon are circled. Numbers in the lighter font refer to the nucleotide position in the JEV genome. (b) Sequence conservation at the P10882 PPMO target site. Alignments of GenBank sequences for four members of the JEV serocomplex in the 3′CSI region are shown. The 3′CSI core sequence location and the nucleotide positions in the JEV genome are indicated below and above the alignment, respectively. Virus abbreviations followed by the GenBank accession number: JEV, Japanese encephalitis virus (NC_001437); WNV, West Nile virus (NC_009942); SLEV, St Louis encephalitis virus (NC_007580); MVEV, Murray Valley encephalitis virus (NC_000943).

Figure 2

PPMO-mediated inhibition of JEV replication in cultured cells. Cell monolayers were treated with 10 µM PPMO for 3 h followed by infection with JEV at an moi of 0.1. Aliquots of culture supernatant were removed at 24 and 36 h p.i. and extracellular virus titrated by plaque assay. All experiments were carried out in triplicate. Mean titres from PPMO-treated cells were compared with those from vehicle-treated cells. The statistical significance of difference in titres was determined by the paired t-test; an asterisk indicates P < 0.05.

Figure 3

Dose-dependent inhibition of JEV replication by the P10882 PPMO. Vero cell monolayers were treated with increasing concentrations of PPMO for 3 h followed by infection with JEV at an moi of 0.1. Scrambled PPMO (PSCR) at a concentration of 10 µM was used as a control. Aliquots of culture supernatant were removed at 24 h p.i. and extracellular virus titrated by plaque assay. All experiments were carried out in triplicate. Mean titres from PPMO-treated cells were compared with those from vehicle-treated cells. The statistical significance of difference in titres was determined by the paired Student's t-test; an asterisk indicates P < 0.05.

Figure 4

PPMO uptake by three types of cultured cells. Cell monolayers were incubated with 10 µM PSCR–Fl PPMO for 3 h followed by washing. The top panels show cells visualized by phase contrast (left-hand panels) and fluorescence microscopy (right-hand panels). In a parallel experiment PSCR–Fl-treated cells were washed and resuspended in culture medium followed by flow cytometric estimation of the fluorescing cells and the fluorescence intensity (bottom left-hand panel) as described in the Materials and methods section. The bottom right-hand panel shows the fold increase in the mean fluorescence intensity (MFI) of cells following treatment with PSCR–Fl. This figure appears in colour in the online version of JAC and in black and white in the printed version of JAC.

Figure 5

PPMO toxicity in mice. Groups of 8-day-old BALB/c mice (n = 8) were injected intracerebrally with a 10–50 mg/kg dose of P10882, as indicated, every 12 h for 8 days and observed for mortality or other signs of toxicity. None of the mice that received up to a 25 mg/kg dose of PPMO showed any apparent signs of toxicity. Survival rates for the various treatment groups are shown by a Kaplan–Meier chart.

Figure 6

PPMO-mediated protection of JEV-infected mice. Groups of 1-week-old BALB/c mice (n = 8) were injected with the indicated amounts of PPMO and infected with 100 pfu of JEV 5 h later. Both PPMO and virus were administered intracerebrally. In addition, mice received the indicated dose of PPMO every 12 h after infection for 5 days. Additional control groups included mice receiving JEV and an initial treatment with vehicle (no PPMO) or uninfected mice receiving P10882 every 12 h for 5 days. Mice mortality was monitored for 10 days. The top three panels show results of the experiment done in triplicate.

Figure 7

JEV titres in mice brain following PPMO treatment. One-week-old BALB/c mice (n = 8) were treated with a 20 mg/kg dose of PPMO as described in the Materials and methods section. Brain tissue was harvested at different timepoints and homogenized for plaque assays. JEV titres in individual mouse brains are shown. The dotted line shows the virus titre assay limit of 50 pfu/mL. Points below the dotted line indicate an undetectable virus titre. Bars indicate the mean titre in a group.