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  1. The effect of apoptotic factors on Sindbis virus infection and replication in the mosquito vector Aedesaegypti upmc-biosecurity.org biochem.ncsu.edu Katelyn O’Neill Graduate Research Forum March 3, 2012

  2. nathnac.org

  3. upmc-biosecurity.org • Sindbis virus is naturally vectored by Culex mosquitoes but can be vectored by Aedes aegypti in the lab • Is not as health threatening as dengue virus • Is well characterized molecularly nathnac.org biochem.ncsu.edu Aedes aegypti also vectors yellow fever virus Has a wide global range Prefers to feed on humans Feeds primarily during the day

  4. Gusmao et al. Mem. Inst. Oswaldo Cruz vol.102 no.8 Rio de Janeiro Dec. 2007

  5. Viruses and Apoptosis:To Kill or Be Killed Apoptosis is often induced in infected cells due to the presence of viral proteins and/or stress on the cell due to viral replication. Apoptosis can be helpful or harmful depending on the virus.

  6. Hypothesis: Inducing or inhibiting apoptosis will have an effect on Sindbis virus replication and spread in the mosquito.

  7. Pro-apoptotic or anti-apoptotic gene Test this by over-expressing anti-apoptotic and pro-apoptotic genes in the mosquito using recombinant Sindbis virus clones Control, same size insert, not coding for a protein Pro-apoptotic gene Anti-apoptotic gene Reaper P35 Wang H., J Gen Virol. 2008 November ; 89(Pt 11): 2651–2661

  8. Looking at infection in mosquito by orally feeding infectious virus clones during blood meal Hemotek Feeding System, Discovery Workshop

  9. Midgut staining TCID50 Assay Dissect α-Sindbis E2 Antibody Stain Triturate Titer in BHK cells

  10. Midgut staining Dissect α-Sindbis E2 Antibody Stain

  11. Antibody staining of midguts from mosquitoes fed a high titer blood meal 3dpi 5dpi 7dpi Rpr sense Rpr antisense (control)

  12. Infection with P35 expressing Sindbis: Midgut Infection Scores 3dpi 5dpi 7dpi p = 0.062 p = 0.7 p = 0.39 n=58 n=44 n=50 n=54 n=56 n=41 Infection score = (surface area of midgut that is infected) x (brightness of antibody stain fluorescence) P35 expression does not have an effect on infection in the midgut p values determined using Mann-Whitney U test

  13. Infection with Reaper expressing Sindbis: Midgut Infection Scores Reaper expression results in less infection in the midgut at early stages of infection 3dpi 5dpi 7dpi p <0.0001 p = 0.0411 p = 0.5084 n=64 n=67 n=52 n=70 n=61 n=55 p values determined using Mann-Whitney U test

  14. Titering with individual mosquitoes Triturate Titer in BHK cells

  15. Infection with P35 expressing Sindbis: Individual Mosquito Titers P35 expression does not have an effect on infection and dissemination 3dpi 5dpi 7dpi p = 0.6514 p = 0.702 p = 0.06 n=33 n=34 n=44 n=43 n=45 n=42 p values determined using Mann-Whitney U test

  16. A possible hypothesis is that these lower titers represent mosquitoes in which the virus does not escape the midgut Infection with Reaper expressing Sindbis: Individual Mosquito Titers 7dpi 3dpi 5dpi p = 0.8087 p = 0.0192 p = 0.7606 n=28 n=35 n=40 n=57 n=39 n=34 Reaper expression lowers infection and slows down dissemination in the mosquito p values determined using Mann-Whitney U test

  17. To test this midguts were titered separately from the rest of the body Dissect Triturate Titer in BHK cells

  18. Midgut vs. Body titered separately 7dpi 3dpi 5dpi p = 0.159 p = 0.03 p = 0.053 p = 0.359 p = 0.374 p = 0.22 p = 0.36 p = 0.558 p = 0.34 n= 31 37 n= 34 36 n=37 36 Body Midgut Body + Midgut Body Midgut Body + Midgut Body Midgut Body + Midgut p values determined using Mann-Whitney U test

  19. Conclusions • Reaper expression • Slows down virus replication in the midgut • Decreases infection and/or dissemination within the mosquito • Possibly due to low initial infection of the midgut and/or decreased midgut escape • P35 expression • Does not have an effect on midgut infection and dissemination within the mosquito

  20. Future Directions Longevity assays Saliva titers Virus competition assays Intrathoracic injection of virus to bypass midgut Characterize other pro-apoptotic clones to see if they have similar effects as rpr clone

  21. Acknowledgements Carol Blair and Ken Olson labs from CSU Passarelli Lab Committee Members Lorena Passarelli Kristin Michel Anna Zolkiewska Clem Lab Rollie Clem Hua Wang Casey Devore Taryn Gort Paula Kurtz Ariel Huang Binny Bhandary John Nail