Bifunctional siRNA containing immunostimulatory motif enhances protection against pandemic H1N1 virus infection.
BACKGROUND
The first influenza pandemic of 21(st) 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.


RESULTS
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.
Gaurav
G
Joshi
Paban Kumar
PK
Dash
Ankita
A
Agarwal
Shashi
S
Sharma
Division of Virology, Defence R&D Establishment (DRDE), Jhansi Road, Gwalior-474002, M.P., India. paridamm@rediffmail.com.
Manmohan
M
Parida
D000276:Adjuvants, Immunologic / Q000494:pharmacology*; D000998:Antiviral Agents / Q000276:immunology*; D017209:Apoptosis / Q000187:drug effects / Q000276:immunology; D002460:Cell Line; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000187:drug effects* / Q000276:immunology*; D007251:Influenza, Human / Q000188:drug therapy* / Q000276:immunology*; D050839:Interferon Regulatory Factor-7 / Q000276:immunology; D016898:Interferon-alpha / Q000276:immunology; D053594:Myeloid Differentiation Factor 88 / Q000276:immunology; D019251:Nucleocapsid / Q000235:genetics; D012333:RNA, Messenger / Q000276:immunology; D034741:RNA, Small Interfering / Q000276:immunology*; D051199:Toll-Like Receptor 7 / Q000276:immunology; D015854:Up-Regulation / Q000187:drug effects / Q000276:immunology
D000276:Adjuvants, Immunologic; D000998:Antiviral Agents; D050839:Interferon Regulatory Factor-7; D016898:Interferon-alpha; C507779:MYD88 protein, human; D053594:Myeloid Differentiation Factor 88; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C495348:TLR7 protein, human; D051199:Toll-Like Receptor 7