Rationale: The first influenza pandemic of 21st century was attributed to a novel quadruple
reassortant H1N1 virus that emerged in 2009. Currently available therapies for influenza have drugresistant.
Therefore, there is a need to develop new generation immunotherapeutic antiviral strategy.
This study described the efficacy of a novel bifunctional immunostimulatory siRNAs against
H1N1pdm swine flu virus by targeting the Nucleocapsid (NP) gene. Methods & Findings: Small interfering
RNAs (siRNA) targeting conserved region of NP were screened for antiviral efficacy in human
lung epithelial cells (A549). Further, a bifunctional siRNA was synthesized by combining immunostimulatory
sequence (5’-UGUGU-3’) with NP specific siRNA. This immunostimulatory siRNA
(NP-1-is) revealed strong antiviral effect through reduction in mRNA copies (99.58%), reduction in virus associated cell
apoptosis and inhibition of nucleocapsid protein in western blot. This immunostimulatory siRNA was found more effective
than nontagged siRNA. Further studies including dose dependent and time course kinetics revealed that the NP-1-is
siRNA is more effective at 20-80 nM with significant protection upto 48 hpi. Besides, the qRT-PCR and western blot
analysis confirmed higher antiviral response of immunostimulatory siRNA was due to upregulation of TLR-7 MyD88,
IRF-7 and IFN-α. Conclusions: This study paves the way for broad-spectrum RNAi-based therapeutics using immunostimulatory
motif towards improved antiviral effect. Hence this approach will be useful to confront the sudden emergence
of pandemic strains.
Keywords: Antiviral activity, Bifunctional siRNA, Small interfering RNAs (siRNA), Swine flu (H1N1) virus.
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