Enterovirus D68 (EV-D68) represents an emerging human pathogen which was noted to disproportionately affect patients with asthma during a large outbreak that began in the fall of 2014. We hypothesized that evolutionary conserved sequences with in the EV-D68 genome might serve as ideal targets for short-interfering RNAs (siRNAs).
Comparative genomic analysis was utilized to identify phylogenetically conserved regions of the EV-68 genome. Three siRNAs targeting these genomic segments were synthesized. Using an established in vitro, human rhabdomyosarcoma cell based model of EV-D68 infection the impact of siRNA transfection on virus growth was assessed. Absorbance, real-time PCR, plaque assay, endpoint dilution and indirect immunofluorescence were measured to quantify the effects of each siRNA on viral proliferation. Dose escalation and cytotoxicity studies were also performed. ANOVA one-way analysis was used to calculate statistical significance.
An siRNA targeting the viral RNA-dependent RNA polymerase gene showed potent ability to protect cells in vitro from EV-68 mediated cytopathic effect, while non-coding negative control siRNAs had no impact on viral replication (p<0.0001). This siRNA induced peak antiviral activity at single picomolar concentrations. Viral genome copy number was reduced greater than 10 fold in treated wells (p<.0001) when measured by real time PCR. Indirect immunofluorescence demonstrated absence of detectable viral VP2 capsid protein 48 hours post-infection. No cytotoxic effect of the siRNA was detected in transfected cells.
The picornavirus EV-68 is susceptible to in vitro, sequence dependent, siRNA mediated targeting of conserved genomic elements within the viral RNA dependent RNA polymerase gene.