19 July 2011 Richard H. Scheuermann, Ph.D. Department of Pathology

1. Sequence Feature Variant Type and Evolutionary Trajectory Analysis using the Influenza Research Database (IRD) 19 July 2011 Richard H. Scheuermann, Ph.D. Department of Pathology U.T. Southwestern Medical Center

2. Outline • Brief overview of NIAID-Sponsored Influenza Research Database (IRD) • Comprehensive integrated database • Analysis and visualization tools • U.S. NIH-funded, free access, open to all • Developed by a team of research scientists, bioinformaticians and professional software developers • www.fludb.org • www.viprbrc.org for other human viral pathogens • Novel approach to genotype-phenotype association studies – Sequence Feature Variant Type (SFVT) analysis • Evolutionary Trajectory analysis of the pandemic (H1N1) 2009 strain

3. Public Health Impact of Influenza • Seasonal flu epidemics occur yearly during the fall/ winter months and result in 3-5 million cases of severe illness worldwide. • More than 200,000 people are hospitalized each year with seasonal flu-related complications in the U.S. • Approximately 36,000 deaths occur due to seasonal flu each year in the U.S. • Populations at highest risk are children under age 2, adults age 65 and older, and groups with other comorbidities. • Pandemics • 1918 Spanish flu (H1N1); 20 - 100 million deaths • 1957 Asian flu (H2N2); 1 - 1.5 million deaths • 1968 Hong Kong flu (H3N2); 750,000 - 1 million deaths • 2009 Swine origin (H1N1); > 16,000 deaths as of March 2010 Source: World Health Organization - http://www.who.int/mediacentre/factsheets/fs211/en/index.html

4. Influenza Virus Orthomyxoviridae family Negative-strand RNA Segmented Enveloped 8 RNA segments encode 11 proteins Classified based on serology of HA and NA

5. IRD Overview www.fludb.org

7. Search Access to Data www.fludb.org

8. Data Types

9. Core Query Attributes

10. Advanced Query Options

11. Segment search results

12. Analysis and Visualization www.fludb.org

13. Analysis and Visualization Tools

14. Workbench Access www.fludb.org

15. My Private Workbench

20. www.viprbrc.org

21. IRD Summary • Funded by U.S. National Institute of Allergy and Infectious Diseases (NIAID) • Free and open access with no use restrictions • Developed by a team of research scientists, bioinformaticians and professional software developers • Comprehensive collection of public data • Novel derived data, novel analytical tools, unique functions • Integration – Integration – Integration • www.fludb.org • www.viprbrc.org

22. Novel approach to genotype-phenotype association studies – Sequence Feature Variant Type (SFVT) Analysis

23. Limitations to Phylogenetics • Traditional virus phylogenetics focuses on comparative analysis of whole genome/genome segments, and is most useful to understand virus evolution • However, the genetic determinants of important viral phenotypes, e.g. virulence, host range, replication efficiency, immune response evation, etc., are determined by focused functional regions of viral proteins • Therefore, specific genotype-phenotype association can be masked by other evolutionary factors that contribute to traditional phylogenetic analysis

24. SFVT approach Influenza A_NS1_nuclear-export-signal_137(10) Influenza A_NS1_alpha-helix_171(17) VT-1 I F D R L E T L I L VT-2 I F N R L E T L I L VT-3 I F D R L E T IV L VT-4 L F D Q L E T L VS VT-5 I F D R L E N L T L VT-6 I F N R L E A L I L VT-7 I Y D R L E T L I L VT-8 I F D R L E T L V L VT-9 I F D R L E NIVL VT-10 I F E R L E T L I L VT-11 L F D QM E T L VS • Identify regions of protein/gene with known structural or functional properties – Sequence Features (SF) • an alpha-helical region, the binding site for another protein, an enzyme active site, an immune epitope • Determine the extent of sequence variation for each SF by defining each unique sequence as a Variant Type (VT) • High-level, comprehensive grouping of all virus strains by VT membership for each SF independently • Genotype-phenotype association statistical analysis, e.g. genetic determinants of host range, virulence, replication rate

25. SF definition • Based on experimentation reported in the literature and 3D protein structures (PDB records) • Captured by manual curation • Defined by the specific amino acid positions in the polypeptide chain • Annotated with the know structural or functional properties

26. Influenza A Sequence Features as of 18JUL2011 4128 SFs total

27. NS1 Sequence Features

28. SF8 (nuclear export signal)

29. VT for SF8 (nuclear export signal)

30. VT-1 strains

31. Do variations in NS1 sequence featureS influence influenza virus host range?

32. NS1 Sequence Features

33. VT for SF8 (nuclear export signal)

34. VT distribution by host

35. Causes of apparent NS1 VT-associated host range restriction • Virus spread - capability + opportunity • Phenotypic property of the virus – limited capacity • Restricted founder effect – limited opportunity • Restricted spatial-temporal distribution • Sampling bias – assumption of random sampling • Oversampling – avian H5N1 in Asia; 2009 H1N1 • Undersampling – large and domestic cats • Linkage to causative variant

36. VT-11 strains

37. VT for SF8 (nuclear export signal)

38. VT lineages

39. VT-4 lineage

41. VT-4 lineage = B allele/group

42. VT-16 & VT-9 lineages

44. VT-7 lineage

46. Evolutionary Trajectory analysis of the pandemic (H1N1) 2009 strain

47. Phylogenetic Analysis • Evolutionary origin • Select a representative pandemic (H1N1) 2009 sequence from the IRD database • BLAST to identify most similar sequences • Assess phylogenetic relationships

48. Pandemic (H1N1) 2009 selection

49. BLAST Result

50. Segment 1 phylogenetic tree Pandemic (H1N1) 2009 Swine/Ohio/2004 Duck/USA/2000s Human/USA/2007 (seasonal) Swine/USA/1990s