In the name of God

1. In the name of God

2. Summer School Influenza Unit, Pasteur Institute of Iran summer 2013 Summer School

3. siRNAvsInfluenza virus Abbas Jamali, Ph.D Summer School Summer School Summer School Influenza Unit, Pasteur Institute of Iran summer 2011 Influenza Unit, Pasteur Institute of Iran summer 2011

4. RNA interference of influenza virus production by directly targeting mRNA for degradation and indirectly inhibiting all viral RNA transcription

7. Why bother using RNAi to inhibit influenza production? • 10-20% of the U.S. population is infected each year resulting in 40,000 deaths • Influenza is easily spread • Antigenic drift (changes in HA and NA- viral antigens) prevents immunity • Antigenic shift (mixing of viruses from 2 different species) creates new strains that also prevent immunity

8. Brief Review of Influenza A • 8 segments of ssRNA- Segments 1-6 each encode 1 protein, 7 & 8 each encode 2 proteins • PB2, PB1, and PA are components of the RNA transcriptase • HA, NA, and NP are the major glycoproteins • Segment 7 encodes M1 and M2 and Segment 8 encodes NS1 and NS2

9. Brief Review of Influenza A Hemagglutinin (HA) is a viral protein anchored in the lipid bilayer that recognizes and binds to host cell’s sialic acid Neuraminidase (NA) is a viral protein anchored in the lipid bilayer that after infection, cleaves off sialic acid from the host cell to prevent “recapturing” of the newly created virions

10. Designing siRNAs specific for Influenza A virus • Remember: RNAi uses dsRNA to direct sequence- specific degradation of mRNA • Search for conserved sequences of 21 nucleotides (any longer sequence would trigger an interferon response) • There are 15 HA subtypes and 9 NA subtypes, none of which have 21 n’tide conserved sequences • Thus, Ge et al. designed 20 different siRNAs specific for NP, PA, PB1, PB2, M, and NS genes and tested them

11. Testing for Influenza Virus Production • 2 testing methods were used: • Inhibition of Flu production in cell lines • Madin-Darby canine kidney (MDCK) cells were used • siRNAs were introduced into the cell and then either A/PR/8/34 (PR8) virus WSN/33 (WSN) virus • Inhibition of Flu production in Embryonated Chicken Eggs • 10-day-old embryonated chicken eggs were used • PR8 virus was introduced alone or in conjunction with the siRNAs • Fifth level

12. Inhibition of Influenza Production in Cell Lines • different siRNAs were introduced into the MDCK cells. 8 hours later, PR8 or WSN virus was added to the cell with a multiplicity of infection (moi) of 0.001, 0.01, or 0.1. • Controls: 1. GFP-949 (siRNA specific for GFP) was introduced into MDCK cells expressing GFP. Later the cells were infected by the viruses. 2. Mock transfection: The virus was introduced into cells with no siRNA • Using an HA assay, the virus titer was determined at different times after the infection for the controls and the tested cells

13. Results of siRNAs in Cell Lines infected with Influenza • Figure A graphically represents the time vs. the virus amount for both PR8 and WSN virus • Mock transfection virus titers increased over time • GFP-949 did not affect virus production – this means that siRNA does NOT interfere nonspecifically with flu virus production

14. Results of siRNAs in Cell Lines infected with Influenza • Together, Figures A and D show 3 different types of results • Approx. 45% of the siRNAs had no effect on the virus titer • Approx. 40% of the siRNAs significantly inhibited virus production • Approx. 15% of the siRNAs potently inhibited production ***NP-1496 and PA-2087 produced no detectable HA activity

15. Results of siRNAs in Cell Lines infected with Influenza • Figure B shows the potentcy of ofsiRNA- the virus titer was determined when MDCK cells were transfected with different concentrations of NP-1496 siRNA • As the amount of siRNA decreased, virus titer increased but still very potent

16. Results of siRNAs in Cell Lines infected with Influenza • Figure C shows that the procedure also works when reversed • First the MDCK cells were infected by the virus and then by the siRNA • Virus titer increased steadily with mock transfection but NP-1496 siRNA worked to keep the virus titer levels low

17. Summary of Cell Line Results • Certain siRNAs potently inhibit flu production in MDCK cells • Influenza production is inhibited by siRNAs specific for different viral genes (notably NP, PA, and PB1) • siRNA works in cells with ongoing infection

18. Testing for Influenza Virus Production • 2 testing methods were used: • Inhibition of Flu production in cell lines • Madin-Darby canine kidney (MDCK) cells were used • siRNAs were introduced into the cell and then either A/PR/8/34 (PR8) virus WSN/33 (WSN) virus • Inhibition of Flu production in Embryonated Chicken Eggs • 10-day-old embryonated chicken eggs were used • PR8 virus was introduced alone or in conjunction with the siRNAs • Fifth level

19. Effects of siRNAs on Virus titer • Mock transfection and GFP-949 had no effect on virus production • The same siRNAs that potently inhibited virus production in MDCK cells (PB1-2257, PA-2087, NP-1496) considerably reduced virus titers in chicken embryos

20. Summary of Important Findings • siRNAs potently inhibit influenza virus production in both cell lines and embryonated chicken eggs • siRNAs that target NP and PA are the most effective • Some siRNAs exert their inhibitory effect by interfering with the accumulation of mRNA AND other viral RNAs (I.e. NP and PA are required for virus replication and translation) • Viral mRNA is the direct target of siRNA-mediated interference

21. Implications • The result of these analyses provide the beginning of exploration of siRNAs’ possible role in an eventual therapy to treat influenza • Epithelial cells of the respiratory tract are affected by the virus and therefore siRNAs could possibly be administered intranasally or pulmonarally