Abstract
BackgroundEnterovirus 71 (EV71) is a highly infectious agent that plays an etiological role in hand, foot, and mouth disease. It is associated with severe neurological complications and has caused significant mortalities in recent large-scale outbreaks. Currently, no effective vaccine or specific clinical therapy is available against EV71.MethodsUnmodified 21 nucleotide small interfering RNAs (siRNAs) and classic 2′-modified (2′-O-methylation or 2′-fluoro modification) siRNAs were designed to target highly conserved 5′ untranslated region (UTR) of the EV71 genome and employed as anti-EV71 agents. Real-time TaqMan RT-PCR, western blot analysis and plaque assays were carried out to evaluate specific viral inhibition by the siRNAs.ResultsTransfection of rhabdomyosarcoma (RD) cells with siRNAs targeting the EV71 genomic 5′ UTR significantly delayed and alleviated the cytopathic effects of EV71 infection, increased cell viability in EV71-infected RD cells. The inhibitory effect on EV71 replication was sequence-specific and dosage-dependent, with significant corresponding decreases in viral RNA, VP1 protein and viral titer. Appropriate 2′-modified siRNAs exhibited similar RNA interference (RNAi) activity with dramatically increased serum stability in comparison with unmodified counterparts.ConclusionSequences were identified within the highly conserved 5′ UTR that can be targeted to effectively inhibit EV71 replication through RNAi strategies. Appropriate 2′-modified siRNAs provide a promising approach to optimizing siRNAs to overcome barriers on RNAi-based antiviral therapies for broader administration.
Highlights
Enterovirus 71 (EV71) is a highly infectious agent that plays an etiological role in hand, foot, and mouth disease
The RD cells were first infected with 0.01 multiplicity of infection (MOI) of EV71 for 1 h, followed by treatment with various small interfering RNAs (siRNAs)
When compared with EV71 China strain genomes available in GenBank in the previous 5 years, siRNAs targeting the 115–133 nt sequence of the EV71 genomic 50 untranslated region (UTR) indicated the rates of sequences with complete nucleotide coincidence reached about 71.30%, while the siRNAs targeting the 648–666 nt sequence of the EV71 genomic 50 UTR showed the rates of sequences with complete nucleotide coincidence (37.04%) and those with the same single-nucleotide difference (41.66%) together reached about 79.70% (Additional file 2: Table S1)
Summary
Enterovirus 71 (EV71) is a highly infectious agent that plays an etiological role in hand, foot, and mouth disease. It is associated with severe neurological complications and has caused significant mortalities in recent large-scale outbreaks. Methods: Unmodified 21 nucleotide small interfering RNAs (siRNAs) and classic 20-modified (20-O-methylation or 20-fluoro modification) siRNAs were designed to target highly conserved 50 untranslated region (UTR) of the EV71 genome and employed as anti-EV71 agents. Conclusion: Sequences were identified within the highly conserved 50 UTR that can be targeted to effectively inhibit EV71 replication through RNAi strategies. Several RNAi-based antiviral therapeutics are currently in various phases of clinical trials for the treatment of viral infections, such as the human immunodeficiency virus, respiratory syncytial virus, and hepatitis C virus [14]. The destruction of EV71 RNA could eliminate protein synthesis and viral replication
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