1. Ferret upper respiratory tract microbiome in influenza infection Barb Methe & Elodie Ghedin, JCVI Adolfo Garcia-Sastre, Mt Sinai, NYC

2. Questions study would address Following Influenza viral infection of the host, how is the bacterial community structure affected? Can we see a change in the bacterial community during the course of the infcetion? Once the host recovers from viral infection, how resilient is the bacterial community structure?

3. Influenza Genome Project

4. Pilot to determine best conditions 2 uninfected (ferrets “94” and “96”) – Nasal swab versus nasal wash – Day 0 and Day 2 – Total specimens: 8 2 infected (ferrets “01” and “08”) + 1 uninfected control (“06”) – Nasal washes only – Day 0, Day 2, Day 3 – Total specimens: 9

5. Replicates and controls Total of 17 specimens – 3 technical replicates of 16S amplicons – 2 regions for each: V1-V3 and V3-V5 – For each round of PCR, 1 positive and 1 negative control Total amplicons (test + controls): 114 Sequencing: average of 4175 classifiable reads per sample

6. STUDY 1 Infected versus uninfected ferrets

7. Infected V1-V3

8. Infected V3-V5

9. Fusobacterium Porphyromonas Control uninfected ferret: Day 0

10. Ureaplasma Cloacibacterium Control uninfected ferret: Day 2

11. Ureaplasma Exiguobacterium Control uninfected ferret: Day 3

12. V1-V3

13. V3-V5 Bacteria rank differently depending on region studied

14. Ferret “01” Ferret “08” Infected ferrets: Day 0

15. Ferret “01” Ferret “08” Infected ferrets: Day 2

16. Ferret “01” Ferret “08” Infected ferrets: Day 3

17. V1-V3

18. V3-V5

19. Summary Important differences depending on region analyzed (V1-V3 vs V3-V5) Ferret effect

20. STUDY 2 Nasal Swab versus Wash

22. Wash captures more species than swab

23. V1-V3 vs V3-V5: Some species overlap and many unique captured by each region

24. Conclusions Based on the results we have obtained thus far, the nasal washes are recovering the greatest diversity using either 16S variable region and are likely to be the most useful samples going forward. Not surprisingly, each 16S variable region returns a slightly different result and one can argue for either region. The V1V3 in general performs better in the lab and it is the region of the 16S gene that is evolving faster than the remainder of the gene (so in theory more discrimination).

25. Teams JCVI Barb Methe Elodie Ghedin Kelvin Li Monika Bihan Sana Scherbakova Mt. Sinai Adolfo Garcia- Sastre Rafael Medina Funding: NIAID

26. Infected V1-V3 V3-V5

27. Uninfected