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

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 – – 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

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); million deaths – 1957 Asian flu (H2N2); million deaths – 1968 Hong Kong flu (H3N2); 750, million deaths – 2009 Swine origin (H1N1); > 16,000 deaths as of March 2010 Source: World Health Organization -

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

IRD Overview

Search Access to Data

Data Types

Core Query Attributes

Advanced Query Options

Segment search results

Analysis and Visualization

Analysis and Visualization Tools

Workbench Access

My Private Workbench

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

NOVEL APPROACH TO GENOTYPE-PHENOTYPE ASSOCIATION STUDIES – SEQUENCE FEATURE VARIANT TYPE (SFVT) ANALYSIS

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

SFVT approach VT-1I F D R L E T L I L VT-2I F N R L E T L I L VT-3I F D R L E T I V L VT-4L F D Q L E T L V S VT-5I F D R L E N L T L VT-6I F N R L E A L I L VT-7I Y D R L E T L I L VT-8I F D R L E T L V L VT-9I F D R L E N I V L VT-10I F E R L E T L I L VT-11 L F D Q M E T L V S Influenza A_NS1_nuclear-export-signal_137(10) 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 Influenza A_NS1_alpha-helix_171(17)

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

Influenza A Sequence Features as of 18JUL SFs total

NS1 Sequence Features

SF8 (nuclear export signal)

VT for SF8 (nuclear export signal)

VT-1 strains

DO VARIATIONS IN NS1 SEQUENCE FEATURES INFLUENCE INFLUENZA VIRUS HOST RANGE?

NS1 Sequence Features

VT for SF8 (nuclear export signal)

VT distribution by host

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

VT-11 strains

VT for SF8 (nuclear export signal)

VT lineages

VT-4 lineage

VT-4 lineage = B allele/group

VT-16 & VT-9 lineages

VT-7 lineage

EVOLUTIONARY TRAJECTORY ANALYSIS OF THE PANDEMIC (H1N1) 2009 STRAIN

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

Pandemic (H1N1) 2009 selection

BLAST Result

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

Temporal component Reference strain – A/California/04/2009 BLAST – Return top 1000 results Normalize data Graph nucleotide differences versus isolation year differences

NP chart

NS chart

HA chart

Group 1 Group 3 Group 2

<= Cali/04/09 NS blue cluster (G1)

<= Cali/04/09 NS green cluster (G2)

Phylogenetic Trees Quantification Analysis method – Build tree for Group 1 and Group 2 strains separately – Analyze branch lengths of trees Results – Avg. Group 1 Branch Length: (S.D ) – Avg. Group 2 Branch Length: (S.D ) – T-test (2 sample, unequal variance):

Group 1 Group 3 Group 2

HA trendline

Evolutionary Trajectory Slopes vs. Mutation Rate SegmentGroup 1 SlopeGroup 2 SlopeMutation Rate PB PB PA HA NP NA M NS Substitutions/segment/year

Evolutionary Trajectory (E.T.) Similar but Distantly Related (SDR)

Garten, et al. Science 2009

Garten, et al. Science 2009

<= Cali/04/09 ET

<= Cali/04/09 SDR

North American H1N1 Lineage - HA H1N American Swine, 2000’s North American H1N1 Lineage HA – Group 1 American Swine, 90’s American Swine, 80’s American Swine, 70’s American Swine, ’s

Evolutionary Trajectory Plots Evolutionary Trajectory of a strain, with candidates displayed.

Summary The Influenza Research Database (IRD) provides a comprehensive resource of data, analysis and visualization tools about influenza virus – SFVT represents a novel tool that can be used to better understand genotype-phenotype relationships for flu Use of IRD to illuminate the viral origins of the pandemic (H1N1) 2009 virus IRD is continually evolving to capture and integrate addition data and analytical tools to support the needs of the influenza research community

72 U.T. Southwestern – Richard Scheuermann (PI) – Burke Squires – Jyothi Noronha – Victoria Hunt – Shubhada Godbole – Brett Pickett – Yun Zhang MSSM – Adolfo Garcia-Sastre – Eric Bortz – Gina Conenello – Peter Palese Vecna – Chris Larsen – Al Ramsey LANL – Catherine Macken – Mira Dimitrijevic U.C. Davis – Nicole Baumgarth Northrop Grumman – Ed Klem – Mike Atassi – Kevin Biersack – Jon Dietrich – Wenjie Hua – Wei Jen – Sanjeev Kumar – Xiaomei Li – Zaigang Liu – Jason Lucas – Michelle Lu – Bruce Quesenberry – Barbara Rotchford – Hongbo Su – Bryan Walters – Jianjun Wang – Sam Zaremba – Liwei Zhou IRD SWG – Gillian Air, OMRF – Carol Cardona, Univ. Minnesota – Adolfo Garcia-Sastre, Mt Sinai – Elodie Ghedin, Univ. Pittsburgh – Martha Nelson, Fogarty – Daniel Perez, Univ. Maryland – Gavin Smith, Duke Singapore – David Spiro, JCVI – Dave Stallknecht, Univ. Georgia – David Topham, Rochester – Richard Webby, St Jude USDA – David Suarez Sage Analytica – Robert Taylor – Lone Simonsen CEIRS Centers Acknowledgments N01AI40041

Segment 6 (NA) By Host