Susceptibility of Enterovirus-D68 to RNAi-mediated antiviral knockdown

Enterovirus D68 (EV-D68) represents an emerging pathogen which has demonstrated a capacity for causing epidemic illness in pediatric and immunocompromised patients. With no effective antiviral treatment available, therapeutic interventions are currently limited to supportive care. Utilizing available genomic sequences from the 2014 B3 Epidemic EV-D68 clade and the 1962 Fermon EV-D68 strains, we performed in silico comparative genomic analysis, identifying several islands of phylogenetic conservation within the viral RNA-dependent RNA polymerase gene. The effects of transfecting short-interfering double-stranded RNA (siRNA) molecules targeting these conserved sequences were tested in vitro using a human rhabdomyosarcoma cell-based model of EV-D68 infection. Two siRNA sequences demonstrated reproducible ability to abrogate EV-D68-mediated cytopathic effect in vitro. These siRNA sequences were also able to decrease EV-D68 genome replication, VP-2 capsid protein expression, and infectious particle production in vitro. EV-D68 knockdown was sequence-specific and not observed in cells treated with a negative control siRNA lacking sequence homology to the viral genome. The regions targeted by these siRNA's are located in highly conserved regions of the RNA-dependent RNA polymerase gene. The most potent siRNA targeted a sequence found in subsequent enzyme crystallographic studies to enhance the enzyme's thermostability (Wang et al., 2017). Topical nebulized siRNAs have recently been utilized as antivirals in human studies, with no adverse effects or toxicities noted (Gottlieb et al., 2016). Sequence selection is likely one primary factor determining the potential efficacy of such therapeutics. These results demonstrate that the identified siRNA sequences are able to suppress EV-D68 replication and cytopathic effect in vitro.